Teneurin c-terminal associated peptides (tcap) and methods and uses thereof

ABSTRACT

The invention provides a novel family of biologically active neuropeptides and the nucleic aid molecules coding for same. The peptides are derived for the C-terminus of the teneurin family peptides (Ten M1-4). These novel peptides, referred to as teneurin C-terminal associated peptides (TCAPs) are active in neuronal communication and are implicated in a number of neuropathologies. They are particularly useful in modulating stress responses and anxiety and in the treatment of cancer.

RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.10/510,959, with a 371 date of Aug. 10, 2005 entitled “TeneurinC-Terminal Associated Peptides (TCAP) and Methods and Uses Thereof”, andwhich is a National Phase entry application of PCT/CA2003/000622 filedMay 2, 2003, which claims the benefit and priority of United Statesprovisional patent application number, U.S. 60/377,231, filed May 3,2002, entitled “Teneurin C-Terminal Associated Peptides (TCAP)” and U.S.60/424,016, filed Nov. 6, 2002, entitled “Method for Modulating Stressusing Teneurin C-Terminal Associated Peptide-1 (TCAP-1)”. Thisapplication also claims priority from United States provisional patentapplication number, U.S. 60/376,879, filed May 2, 2002, entitled,“Immortalized Hypothalamic Neuronal Cell Lines”. All of these referencesare incorporated in their entirety by reference.

FIELD OF THE INVENTION

The invention relates to a novel family of peptides associated with thec-terminal region of the teneurin molecule, to a nucleic acid moleculeencoding said peptides and to methods and uses therefore.

BACKGROUND OF THE INVENTION

The aetiology of any neuropathology is a complex interplay of genetic,physiological and environmental factors. Effective treatment of theseconditions will ultimately depend upon the understanding of the cognategenes and their products. In recent years, it has become apparent thatlarge families of related genes are responsible for the regulation ofneuropathologies involving anxiogenic peptides. The identification andcharacterization of these gene families and how they interact is anessential step towards ultimately effectively treating the pathology.The aberrant regulation of neuronal growth can manifest as a variety ofpathological conditions depending upon the age. Deficits in neuronalgrowth in foetal or neonatal animals can cause such diseases as learningdeficits, mental retardation, autism, or schizophrenia. At later ages injuvenile individuals it may manifest as affective disorders such aspanic disorder, depression, anorexia nervosa, obsessive-compulsivedisorder later in adults. In adults such neuronal growth problems couldlead to neurodegenerative illnesses such as Alzheimer's Disease orParkinsons's Disease.

The onset of mood disorders, such as depression or post traumatic stressdisorder, involve the altered function of multiple loci in the brainthat regulate emotionality, memory and motivation (Manji et al., 2001;Drevets, 2001; Nestler et al., 2002). However, many of the cellularsignaling molecules that mediate communication within and between theseregions are unknown, leading to an incomplete understanding of theorigin of such disorders.

Many neuropeptides show the presence of three or four paralogousstructures as evidenced by the neuropeptide Y (NPY) (Larhammar,1996a,b), proopiomelanocortin (POMC) (Danielson, 2000) and recently, thecorticotropin releasing factor (CRF) family (Vale et al., 1981, Vaughanet al., 1995; Lovejoy and Balment, 1999; Lewis et al., 2001 Reyes etal., 2001; Hsu and Hseuh, 2001).

A family of neuronal cell surface proteins has been identified that arepredominantly expressed in the nervous system. These proteins have beennamed teneurins (Rubin et al, Developmental Biology 216, 195-209(1999)). Four basic teneurins have been identified Ten M1, Ten M2, TenM3, and Ten M4. The Ten-M or Odz proteins were originally discovered inDrosophilia (Levine et al., 1994; Baumgartner et al., 1994) and arepresently the only known example of a pair-rule gene that is not atranscription factor. The Ten-M gene is initially activated during theblastoderm stage, then down regulated before being expressed at laterstages. The highest levels of Ten-M occur in the central nervous systemwhere the protein occurs preferentially on the surface of axons (Levineet al., 1994; Levine et al, 1997). Mutations of the Ten-M/Odz generesult in embryonic lethality (Baumgartner et al., 1994; Levine et al.,1994).

Four Ten-M paralogous genes, called Teneurins, exist in vertebrates andencode a Type II transmembrane protein where the carboxy terminus of theprotein is displayed on the extracellular face of the cell (Oohashi etal., 1999). The teneurin proteins are about 2800 amino acids long. Thereis a short stretch of hydrophobic residues at 300 to 400 amino acidsafter the amino terminus that appear to act as the membrane spanningsite. In the cytoplasmic N-terminal portion, is a conserved proline-richSH3-binding site indicating a potential site where by they bind otherproteins. Evidence suggests that the protein may be cleaved from themembrane at a Furin-like cleavage motif (RERR) located around residue528 in teneurin 2 (Rubin et al., 1999). However, this motif is notpresent in the other paralogues and therefore a soluble version of theprotein may not occur for all paralogues. There are a series ofcysteine-rich EGF-like repeats carboxy terminal to this.Homodimerization occurs between Ten M1 forms via interaction betweenEGF-like modules 2 and 5 (Oohashi et al., 1999).

The ten-m gene appears to be upregulated by stressors. Wang et al (1998)showed that a ten-M like transcript, named DOC4 (downstream of chop) inmammalian cells was upregulated by the transcription factorGADD153/CHOP. This transcription factor is induced by several types ofcellular stressors including UV light, alkylating agents or conditionstriggering endoplasmic reticulum (ER) stress responses, such as,deprivation of oxygen, glucose or amino acids, or interference ofcalcium flux across the ER membrane (Zinszner et al, 1998). GADD153 is asmall nuclear protein that dimerizes with members of the C/EBP family oftranscription factors (Ron and Habener, 1992). It does not appear tohomodimerize. GADD153 undergoes a stressor inducible phosphorylation bya p38-type MAP kinase which also enhances the transcriptional activationof GADD153 (Wang et al., 1996). High expressions of GADD153 will lead tocell cycle arrest (Zhan et al. 1994). These studies suggest that theteneurin gene may play a significant role in the regulation of thestress response of neurons and other cells.

Overexpression of teneurin 2 into the mouse neuroblastoma cells (Nb2a)augmented the amount of neurite outgrowth and a tendency to enlarge thegrowth cones. The number of filamentous actin-containing filopodia wasalso enhanced in the teneurin 2 overexpressing cells (Rubin et al.,1999). The expression of the teneurin genes have been examined inembryonic zebrafish (Mieda et al, 1999), chicken (Rubin et al., 1999)and mouse (Ben-Zur et al., 2000) although their expression patterns havenot been finely resolved. The transcripts are found in a number ofperipheral tissues but are found predominantly in the central nervoussystem. In the embryonic chicken brain, teneurin 1 and 2 are expressedin the retina, telencephalon, the optic tectum and the diencephalons.The mRNA for teneurin 1 was found mainly in the intermediate zone of thedorsal thalamus whereas teneurin 2 was found in the intermediate zone ofthe thalamus (Rubin et al., 1999). In zebrafish, teneurin 4 is faintlyexpressed throughout gastrulation, although there is no teneurin 3expression. Teneurin 3 expression begins at the notochord and the somitearound the tailbud stage. In later stages (14 h post fertilization),teneurin 3 is expressed in the somites, notochord and brain whileteneurin 4 expression was confined to the brain. Teneurin 3 becomesdefined within the optic vesicles and region covering the caudaldiencephalons and mesencephalon with the expression strongest in theanterior mesencephalon. Teneurin 4 has its strongest expression towardthe midbrain hindbrain border. By 23 h post fertilization, teneurin 3 isexpressed in the dorsal part of the tectal primordium and the ventralmidbrain while teneurin 4 is expressed in the ventral primordium (Miedaet al., 1999).

Neuropathological conditions tend to be complex and not very wellunderstood. As such, there is a need to better understand the mechanismsinvolved and to develop a method of diagnosis and treatment of saidconditions. There is also a need for the identification and design oftherapeutic compounds for said conditions.

SUMMARY OF THE INVENTION

The present invention provides a teneurin c-terminal associated peptide(TCAP), existing as a 40-41-residue sequence on the c-terminal exon ofTen-M 1, 2, 3, or 4 that is correspondingly named TCAP 1, 2, 3, and 4.In another embodiment, the invention provides a peptide that has theamino acid sequence consisting of a 40- or 41 amino acid sequencelocated at the c-terminus of the teneurin 1-4 peptides, to analogs,species homologues, dervivatives, variants, allelic variants, tosequences having substantial sequence identity thereto and to obviouschemical equivalents thereto. In another embodiment the TCAP peptides ofthe invention can further include an amidation signal sequence at thecarboxy terminus (hereinafter referred to as “preTCAP”). Such amidationsignal amino acid sequence can include but is not limited to GKR andGRR. The invention also provides fusion proteins comprising the TCAPpeptides noted above, to labeled TCAP Peptides and to peptidescomprising flanking amino acid sequence of 1-10 amino acids.

In one embodiment, the invention provides a TCAP peptide that hasneuronal communication activity. In another embodiment the inventionprovides a TCAP peptide, an analog, derivative, variant, homolog thathas similar activity. In one embodiment, the activity is neuronalcommunication. In another embodiment it is inhibition of cellproliferation. In yet another embodiment it is modulation of a stressresponse.

In one embodiment the TCAP sequence is a rainbow trout, zebrafish,human, mouse, G. gafius, or D. melanogaster TCAP. In another embodiment,the TCAP sequence comprises or consists of SEQ. ID. NOS: 13, 14, 21, 22,29, 30, 37, 38, 45, 46, 53, 54, 61, 62, 69, 70, 77, 78, 85, 86, 93, 94,101, 103. In yet another embodiment, the TCAP is a mouse or human TCAP.In one embodiment the TCAP has one of the sequences selected from thegroup consisting of SEQ. ID. NOS: 69, 70, 77, 78, 85, 86, 93, 94 (human)or SEQ. ID. NOS: 37, 38, 45, 46, 53, 54, 61, 62, (mouse).

In one aspect, the invention provides a TCAP consisting of any one ofthe SEQ. ID. NOS. noted above and an amidation signal sequence at thecarboxy terminus. Preferably the amidation signal sequence is selectedfrom the group consisting of GRR or GKR, such as, 15, 16, 23, 24, 31,32, 39, 40, 47, 48, 55, 56, 63, 64, 71, 72, 79, 80, 87, 88, 95, 96.

Another aspect of the invention relates to an isolated teneurinc-terminal associated peptide that has the amino acid sequence as shownin SEQ. ID. NOS: 13, 14, 21, 22, 29, 30, 37, 38, 45, 46, 53, 54, 61, 62,69, 70, 77, 78, 85, 86, 93, 94, 101, 103; or a fragment, analog,homolog, derivative or mimetic thereof. In a preferred embodiment, theTCAP peptides of the invention have anxiogenic activity. The inventionalso encompasses an antibody that can bind a TCAP peptide of theinvention.

In another embodiment, the peptide of the invention is a TCAP mousepeptide having the amino acid sequence of: SEQ. ID. NOS: 37, 38, 45, 46,53, 54, 61, 62.

In another embodiment, the peptide of the invention is a TCAP humanpeptide having the amino acid sequence of SEQ. ID. NOS: 69, 70, 77, 78,85, 86, 93, or 94.

In another embodiment the peptides TCAP human and mouse peptides have anamidation signal sequence at the C-terminus.

In another embodiment, the peptide of the invention is a TCAP-1 and hasthe amino acid sequence of SEQ. ID. NOS.: 37, 38, 69 or 70.

In another embodiment, the peptide of the invention is a TCAP-2 and hasthe amino acid sequence of SEQ. ID. NOS.: 46, 47, 77, or 78.

In another embodiment, the peptide of the invention is a TCAP-3 and hasthe following amino acid sequence motif:

-   -   QLLSXaa₁Xaa₂ KVXaa₃GYDGYYVLSXaa₄EQYPELADSANNXaa₅QFLRQ SEI (SEQ.        ID. NO:135),        where Xaa₁ is G, S, or A; Xaa₂ is G or R; Xaa₃ is L or Q; Xaa₄        and Xaa₅ are independently V or I. In one embodiment, the TCAP-3        is a human or mouse TCAP-3. In another embodiment, the TCAP-3        has SEQ. ID. NO: 85, 86, 53, or 54. In another embodiment, the        TCAP 3 sequence is SEQ. ID. NO.: 13, 14, 21 or 22.

In another embodiment, the peptide of the invention is a TCAP-4 and hasthe amino acid sequence SEQ. ID. NOS.: 29, 30, 61, 62, 93, or 94.

In another embodiment the peptides TCAP 1 to TCAP 4 have an amidationsignal sequence at the C-terminus.

In yet another embodiment, the present invention provides as isolatednucleic acid molecule encoding a teneurin c-terminal associated peptide(TCAP) of the invention, as noted herein. In yet another embodiment, theisolated nucleic acid molecule of the invention consists of:

(a) a nucleic acid sequence as shown in SEQ. ID. NOS.: 17-20, 25-28,33-36, 41-44, 49-52, 57-60, 65-68, 73-76, 81-84, 89-92, 97-100 or thatwherein T can also be U or that encodes a peptide having an amino acidsequence selected from the group consisting of: SEQ. ID. NOS: 13, 14,21, 22, 29, 30, 37, 38, 45, 46, 53, 54, 61, 62, 69, 70, 77, 78, 85, 86,93, 94, 101, 103 or that further has an amidation signal sequence(preferably GKR or GRR), at the carboxy terminus of said peptides, suchas 15, 16, 23, 24, 31, 32, 39, 40, 47, 48, 55, 56, 63, 64, 71, 72, 79,80, 87, 88, 95, 96;

(b) a nucleic acid sequence that is complimentary to a nucleic acidsequence of (a) or (b);

(c) a nucleic acid sequence that has substantial sequence homology to anucleic acid sequence of (a), or (b);

(d) a nucleic acid sequence that is an analog of a nucleic acid sequenceof (a), (b), or (c); or

(e) a nucleic acid sequence that hybridizes to a nucleic acid sequenceof (a), (b), (c), or (d) under stringent hybridization conditions.

In a preferred embodiment the nucleic acid molecules of the inventionencode teneurin c-terminal associated peptide that has anxiogenicactivity.

The invention also encompasses antisense oligonucleotides complimentaryto a nucleic acid sequence of the invention as well as expressionvectors comprising a nucleic acid molecule of the invention and hostcells transformed with the aforementioned expression vectors.

A further aspect of the invention relates to a method of identifyingsubstances which can bind with a teneurin c-terminal associated peptide,comprising the steps of incubating a teneurin c-terminal associatedpeptide and a test substance, under conditions which allow for formationof a complex between the teneurin c-terminal associated peptide and thetest substance, and assaying for complexes of the teneurin c-terminalassociated peptide and the test substance, for free substance or for noncomplexed teneurin c-terminal associated peptide, wherein the presenceof complexes indicates that the test substance is capable of binding ateneurin c-terminal associated peptide.

The invention also provides a method of identifying a compound thataffects the regulation of neuronal growth comprising incubating a testcompound with a teneurin c-terminal associated peptide or a nucleic acidencoding a teneurin c-terminal associated peptide; and determining anamount of teneurin c-terminal associated peptide protein activity orexpression and comparing with a control, wherein a change in the TCAPpeptide activity or expression as compared to the control indicates thatthe test compound has an effect on the regulation of neuronal growth.

The invention also provides a method of inhibiting cell proliferationcomprising administering to a cell, an effective amount of teneurinc-terminal associated peptide that inhibits cell proliferation. In apreferred embodiment, the inhibited cells are selected from the groupconsisting of neuronal or fibroblast cells.

Another aspect of the invention relates to a method of detecting acondition associated with the aberrant regulation of neuronal growthcomprising assaying a sample for a nucleic acid molecule encoding ateneurin c-terminal associated peptide or a fragment thereof or ateneurin c-terminal associated peptide or a fragment thereof.

The invention also relates to a method of treating a conditionassociated with the aberrant regulation of neuronal growth, for instancecancer, comprising administering to a cell or animal in need thereof, aneffective amount of teneurin c-terminal associated peptide or an agentthat modulates teneurin c-terminal associated peptide expression and/oractivity.

The teneurin-1 mRNA containing the TCAP-1 sequence is expressed inregions of the forebrain and limbic system regulating stress responsesand anxiety. TCAP signals through a specific cAMP-dependentG-protein-coupled receptor to modify cell cycle and proliferation inimmortalized neurons. Administration of synthetic TCAP-1 into thelateral ventricle or amygdala of rats normalized the acoustic startleresponse. These peptides, therefore, appear to be an integral part ofthe neural stress response and likely play a role in the aetiology ofsome psychiatric illnesses.

In another embodiment, the invention provides a method of modulating thestress response in an animal, preferably in a mammal, preferably ahuman, by administering to said animal an effective amount of TCAP,preferably TCAP-1 peptide, a nucleic acid molecule coding for said TCAPpeptide in a form that can express said peptide in situ or an antagonistor agonist of TCAP expression or activity, to modulate the stressresponse in said animal. In one embodiment the stress response is ananxiety response.

In another embodiment, the invention provides a method for normalizingthe stress or anxiety response in an animal. In another embodiment, theinvention provides a method for inducing an anxiogenic response in a lowanxiety animal and for inducing an anxiolytic effect in a high anxietyanimal.

In another embodiment, the invention provides a method modulating thestress response in an animal by modulating the effect of TCAP expressionin an animal by administering to said animal a modulator of said TCAPexpression or activity. In one embodiment said modulator is an inhibitorof TCAP expression and/or activity, in another embodiment, saidmodulator is an antagonist of TCAP expression or activity. In oneembodiment said TCAP is TCAP-1.

In yet another embodiment, said invention provides a method ofdiagnosing an animal with high, normal or low stress response conditionby administering to said animal a TCAP, such as TCAP-1 and monitoringwhether it has an anxiolytic, anxiogenic or neutral effect on a stressresponse of the animal.

Other aspects of the invention relate to methods of inducing ananxiogenic response in a subject, methods of inhibiting damages causedby physiological stresses and methods of inhibiting cell death, eachcomprising administering to a subject an effective amount of teneurinc-terminal associated peptide for affecting the desired result.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 shows a putative 3′ exon of the rainbow trout Teneurin 3 gene[SEQ. ID. NO: 2] with an intron region [SEQ. ID. NO: 1] (1490 bp). Theexon/intron border as established by sequence comparison with the humanten M1 gene (LocusLink ID#10178) shown in the genome database. Theintron placement was subsequently confirmed by PCR. The exon encodes thecarboxy terminal 251 residues of the protein SEQ. ID. NO: 3. Cleavagesignals are indicated in the bolded grey regions. The Terminal GKR motifusually signifies a post translation amidation signal. Theteneurin-associated c-terminal peptide (TCAP) is shown by the sequencebetween amino acids 208 and 248 inclusive [SEQ. ID. NOS: 13 and 14].

FIG. 2 shows the alignment of the amino acid sequences encoded by theterminal exon of the rainbow trout (O. mykiss) SEQ. ID. NO: 3, zebrafish(R. danio) SEQ. ID. NO: 12, mouse (M. musculus) SEQ. ID. NO: 6 and human(H. sapiens) SEQ. ID. NO: 10 genes. All possess an additional serineinsertion in position 58. All show a high sequence similarity with about94% between trout and zebrafish, 83% between rainbow trout and mouse,and 83% between rainbow trout and human. Within the TCAP portion itself,rainbow trout SEQ. ID. NO: 13 or 14 shares 90% sequence identity withzebrafish SEQ. ID. NO: 21 or 22, 90% sequence identity with mouse SEQ.ID. NO:53 or 54, and 88% with human SEQ. ID. NO. 85 or 86. The preTCAPsequences that include the amidation signal are SEQ. ID. NOS: 15-16(Rainbow Trout), 23-24 (zebrafish), 55-56 (mouse) and 87-88 (human).

FIG. 3 shows the alignment of the amino acid sequences encoded by theterminal exon of the mouse teneurin 1, 2, 3 and 4) SEQ. ID. NOS: 4, 5,6, 7 genes. The highest level of sequence similarity occurs among thesequences encoding the TCAP portion of the protein. TCAP-1 SEQ. ID. NO:37 or 38 is 68% identical to TCAP-2 SEQ. ID. NO. 45 or 46, 76% identicalto TCAP-3 SEQ. ID. NO. 53 or 54, and 85% identical to TCAP-4 SEQ. ID.NO. 61 or 62. TCAP-2 is 75% identical with TCAP-3, and 68% identicalwith TCAP-4. TCAP-3 possesses 71% identity with TCAP-4. Teneurin 3possesses a dibasic cleavage site at the amino terminus of TCAP-3whereas 1, 2 and 4 all possess monobasic sites suggesting that thecleaved peptide is 40 residues in TCAP-3 but 41 residues in TCAP-1, 2and 4. However, in one embodiment, both the 41 and 40 amino acid residueTCAP has activity.

FIG. 4 shows the alignment of amino acid sequences encoded by the lastexon of the human Teneurin 1, 2, 3 and 4 proteins SEQ. ID. NOS: 8, 9,10, 11. Like the mouse sequence, the highest degree of sequencesimilarity occurs in the TCAP portion of the exon. TCAP-3 possesses adibasic leaved signal whereas the others possess a monobasic site. Thegreatest variable region occurs with the first 70-80 residues of theexon. Within the TCAP portion itself, TCAP-1 SEQ. ID. NO: 69 or 70shares 73% identity with TCAP-2 SEQ. ID. NO: 77 or 78, 83% identity withTCAP-3 SEQ. ID. NO: 85 or 86 and 88% identity with TCAP-4 SEQ. ID. NO.93 or 94. TCAP-2 has 76% identity with TCAP-3 and 71% identity withTCAP-4. TCAP-3 has 76% identity with TCAP-4.

FIG. 5 shows the nucleotide coding sequence of the preTCAP sequences forHuman (SEQ. ID. NOS: 76, 84, 92, and 100) and Mouse (SEQ. ID. NOS. 44,52, 60 and 68) preTCAP-1 to 4, Zebrafish preTCAP-3 and 4 (SEQ. ID. NOS:28 and 36), and Rainbow Trout preTCAP-3 (SEQ. ID. NO. 20) with stopcodon. The coding region of the corresponding mature TCAP peptides wouldlack the terminal amidation and stop codon coding sequence (e.g. thelast 12 nucleotide bases shown for each sequence). The sequences showncode for the 44 amino acid residue preTCAP sequence with stop codon.However, the 43 amino acid TCAP coding sequence is identical except withthe first three nucleotides absent.

FIG. 6A is a schematic representation of the functional domains withinthe Teneurin protein. FIG. 6B is a schematic view of the exons on humanteneurin 1 and an exploded view of the location of the C-terminal exonand location of TCAP thereon (SEQ ID NO:137). A conserved prohormoneconvertase-like cleavage motif is shown as grey boxes. It illustratesthe structure of Teneurin C-terminal Associated Peptides and theirlocation on the teneurin protein and gene.

FIG. 7A shows the alignment of the human, mouse, rat, chicken, rainbowtrout, zebrafish and drosopholia TCAP sequences SEQ. ID. NOS: 69, 78,85, 94, 37, 46, 53, 66, 78, 101, 136, 13, 21, 30 and 103 and 7B showsthe alignment of the TCAP sequences from mammals birds insects andnematodes FIG. 7B SEQ. ID. NOS: 37, 138, 69, 61, 93, 53, 85, 13, 21, 77,29, and 103. In FIG. 7B, non homologous amino acid substitutions areshaded in light grey. Homologous residues are shaded in dark grey.

FIG. 8 shows the alignment of the amino acid sequences of the human CRFfamily SEQ. ID. NOS: 104-107 with those of the human TCAP family SEQ.ID. NOS: 70, 78, 85, 94. Although overall sequence identity is onlyabout 20-25%, many of the other substitutions reflect potential singlebase codon changes such as proline to serine, leucine or threonine, orconservative amino acid substitutions such as leucine to valine orisoleucine, aspartic acid to glutamic acid and asparagines to glutamine.

FIG. 9 is a comparison of the sequence identity among CRF family membersto that of the identity among TCAP members. The TCAP family members showa much greater sequence identity of 68% compared to the CRF familymembers of 34% between CRF and U3 and U2, 43% between CRF and urocortin,and 21% between urocortin 1 and 3.

FIG. 10 shows a secondary structure prediction of TCAP (Rainbow TroutTCAP-3) and comparison with CRF-like peptides. FIG. 10 A is a GranthamPolarity Prediction and FIG. 10B is a Kyte-Doolittle HydrophobicityPrediction. TCAP shows a highly similar polarity profile, but appears topossess higher levels of total hydrophobicity in the amino terminus.

FIG. 11 shows the alignment of amino acid sequences of representationsof TCAP peptides with the insect diuretic peptides and CRF superfamilySEQ. ID. NOS: 13, 22, 104, 107-110. The entire superfamily can bedivided into three general regions encompassing an amino terminalportion, a midsection and a carboxy terminal portion. All peptides canbe aligned by the presence of conserved motifs within each of theseparate sections

FIG. 12 illustrates expression of Teneurins in mouse brain and celllines NLT, Gn11, and Nero2a. PCR-amplified products corresponding toTeneurin 1 to 4 were found in whole brain and cell lines. TenM1, 2 and 4were found in whole brain and in the immortalized GnRH-expressingneuronal line, Gn11. Only Teneurin 2 and 4 were found in anotherGnRH-expressing cell, NLT, however, all four forms were found in theNeuro2a neuroblastoma cell line. The bands on top indicate positivesignals for the Teneurin transcripts. The bands at the bottom show apositive signal for glyceraldehydes-3-phosphate dehydrogenase (GAPDH) toindicate the viability of the RNA. A 100-bp DNA ladder is shown at theleft of all PCR gels.

FIG. 13 is a bar graph illustrating the inhibition of cell proliferationin Gn11 neuronal cells by 10⁻⁶ M TCAP (Rainbow Trout TCAP-3) at 48 hours(FIG. 13 A) and at 72 hours (FIG. 13B).

FIG. 14 is a bar graph illustrating the inhibition of cell proliferationin TGR1 (wildtype) fibroblast cells.

FIG. 15 is a bar graph illustrating the inhibition of cell proliferationin H016 (c-myc constitutively expressed cells) (14B) by 10⁻⁶ M TCAP(Rainbow Trout TCAP-3) at 48 hours).

FIGS. 16A and 16B are bar graphs illustrating the inhibition of cAMP(16A) and cGMP (16B) accumulation in Gn11 cells by rtTCAP-3 (RainbowTrout TCAP-3). A. 10⁻⁸ M TCAP induced a significant (p<0.01) decrease incAMP concentrations relative to the vehicle-treated cells. Replications:vehicle, n=10; urocortin, n=8; TCAP, n=11. B. 10⁻⁸ M TCAP induced asignificant (p<0.01) decrease in cGMP accumulation in Gn11 cells. Thesame concentration of rat urocortin also induced a significant (p<0.05)decrease in cGMP concentrations. Three replications were used for eachof the treatment groups. Significance was assessed using a one-wayanalysis of variance with a Dunnett's post-hoc test. An a priori levelof significance was established at p=0.05. The original data wastransformed to show percent concentration relative to thevehicle-treated cells.

FIG. 17 A-D illustrates TCAP (Rainbow Trout TCAP-3) cAMP regulation inGn11 cells. 17A illustrates cAMP levels in Gn11 cells treated with 10⁻⁸M TCAP or urocortin over 30 minutes. 17B illustrates cAMP levels in Gn11cells in the presence of 10⁻⁴ M 3-isobutyl-1 methyl xanthine (IBMX), aphosphodiesterase inhibitor used to boost cAMP induced by treatment of10⁻⁸ MTCAP or urocortin. 17C is a bar graph illustrating cAMPaccumulation over 30 minutes in Gn11 cells by administration of variousconcentrations of TCAP or Urocortin in the presence of IBMX. 17D is abar graph illustrating inhibition of 10⁻⁸ M forskolin-stimulated cAMP by10⁻⁸ MTCAP or urocortin.

FIGS. 18A and 18B are linear graphs illustrating the effect of TCAP(Rainbow Trout TCAP-3) on the administration of self reward behaviour.The behaviour was indicated by number of bar presses per 30 seconds overa range of pleasurable stimulation (25-100 Hz). FIG. 18A: Baseline, TCAPpeptide (1.0 μl of 0.001 mg/ml, left), post-injection (approx. 90 min.),850 uA. FIG. 18B: Baseline, TCAP peptide ((1.0 μl of 0.001 mg/ml,right), postinjection (approx. 60 min.), 550 uA. 100 nM TCAP induced asignificant decrease in the rats desire to self-administer reward byneural impulse.

FIG. 19 A schematic cellular model for TCAP regulation. A. A stressor inthe form of a physiological condition such as low oxygen or pH changes,or an anxiogenic ligand triggers metabolic activation of the cell. B.This causes an upregulation of the Teneurin protein and its cleavingenzyme. C. The enzyme liberates TCAP from Teneurin where it acts in anautocrine and paracrine manner to inhibit cAMP and cGMP production via aG protein coupled receptor.

FIG. 20 A-F illustrates the distribution of TCAP-1 mRNA in rat brainnuclei as explained in Example 9.

FIG. 21 are bar graphs illustrating the chronic human TCAP-1 response inrats that were (A) vehicle treated ICV injected, (B) TCAP-1 ICV injectedas described in Example 10 herein.

FIG. 22 are graphs illustrating the mean baseline startle response ofall animals in Example 10. FIG. 22A is the average startle response atday 1 after TCAP injection and FIG. 22B is the average startle at theend of the chronic TCAP study, FIG. 22C is the average startle responsefollowing TCAP-1.

FIG. 23 is the interaction bar plot for treatment with TCAP-1 at variousdoses for both high and low anxiety response animals as discussed inExample 11 herein.

FIG. 24 is the plot of the effect of TCAP-1 amygdala—injected on thestartle response of rats as discussed in Example 11 herein.

FIG. 25 illustrates activity of TCAP on immortalized neurons. (A) cAMPaccumulation in Gn11 cells. 1 nM TCAP increased cAMP (p<0.05) whereas100 nM TCAP decreased (p<0.05) cAMP. An intermediate concentration (10nM) was without effect. (B) Action of CRF-R1 antagonist on cAMPaccumulation. A 1 nM mouse TCAP-1, or mouse urocortin increased cAMPaccumulation in Gn11 cells. The CRF R1 receptor antagonist PD171729abolished the action of urocortin on these cells (p<0.01) but had noeffect on TCAP-mediated cAMP accumulation. (C) Protein assays.Concentrations of 1 to 100 nM TCAP stimulated protein synthesis in Gn11cells. (D) MTT Assay. 1 nM of mouse TCAP-1 increased MU activity(p<0.05) in Gn11 cells after 48 hours. In contrast, 100 nM of mouseTCAP-1 decreased (p<0.05) MTT activity over the same time period. Thelevel of significance was determined using a one-way ANOVA for A and B,and a two-way ANOVA for C and D.

FIG. 26 illustrates the functional cAMP response of murine hypothalamicimmortalized cell lines to TCAP (rainbow trout TCAP-3) peptidestimulation.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have identified a novel peptide sequence which exists aspart of a larger protein previously identified as the Ten M proteins orTeneurins. The novel peptides are referred to as teneurin C terminalpeptides or TCAP. The genomes or gene transcripts of several vertebrateand invertebrate species were screened by homologous probe hybridizationor by PCR. Sequence data from genome sequencing programs allowed theidentification of a complement of four paralogous peptides from thisfamily in humans and mice, two paralogues in zebrafish, one in rainbowtrout and Drosophila (SEQ ID NO:103). The synthetic TCAP peptide hasneuronal communication activity and has been shown to be a modulator ofthe stress response and anxiety in an animal. TCAP also modulates cellproliferation. In one embodiment, it can inhibit cell proliferation. Inanother embodiment, TCAP is a potent anxiogenic peptide in rats andhighly effective at inhibiting neuronal proliferation in unstressedcells and protecting cells from physiological stresses. As such TCAPand/or modulators of TCAP can be used in the treatment of cancer andneuropathological conditions, including those related to neuronalcommunication, and/or cell proliferation, for instance, cancer, stressanxiety, food-related disorders, such as anorexia and/or obesity.

The TCAP sequence encodes a cleavable peptide 40 amino acids longflanked by PC7-like cleavage motifs on the amino terminus and anamidation motif on the carboxy terminus. Depending on the cleavage ofthe PC7-like cleavage site at the N-terminus, the resulting mature TCAPpeptide is 40-41 amino acids in length. The TCAP sequence with thecarboxy terminus amidation motif is herein referred to as preTCAP.Orthologues in humans, mice, zebrafish and Drosophila as well as threeadditional paralogous sequences have been identified. A syntheticversion of the rainbow trout peptide significantly increases the startlereflex and decreases self-administered brain stimulation in rats. Thesefindings are consistent with the actions of peptides known to induceanxiety in mammals and humans. The peptide is also potent at inhibitingthe proliferation of unstressed neuronal and fibroblast cell culturesand inhibiting cell death in these cultures subjected to high pH stress.These findings indicate that TCAP plays a role in the developing andadult brain to modulate and protect neuronal growth and metabolism andtherefore be implicated in a number of pathologies includingschizophrenia, Parkinson's disease and other mental conditions. In theadult brain the peptide may act to modulate the actions of anxiogenicstimuli and could play a role in depression, anorexia nervosa and otheraffective disorders.

The term “isolated” as used herein means “altered by the hand of man”from the natural state. If a composition or substance occurs in nature,the isolated form has been changed or removed from its originalenvironment, or both. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated,” as the term is employed herein. Thus, apolypeptide or polynucleotide produced and/or contained within arecombinant host cell is considered isolated for purposes of the presentinvention. Also intended as an “isolated polypeptide” or an “isolatedpolynucleotide” are polypeptides or polynucleotides that have beenpurified, partially or substantially, from a recombinant host cell orfrom a native source. For example, a recombinantly produced version ofTCAP peptides and derivatives thereof can be substantially purified bymethods known in the art, such as the one-step method described in Smithand Johnson, Gene 67:31-40 (1988).

Nucleic Acid Molecules of the Invention

The present invention provides an isolated nucleic acid moleculeconsisting of a sequence encoding a teneurin c-terminal associatedpeptide. This peptide is generally referred to as “TCAP” herein. Thepresent invention also provides an isolated nucleic acid moleculeencoding a TCAP peptide with a carboxy terminus amidation motif, saidpeptide herein referred to as “preTCAP”.

Isolated nucleic acids substantially free of cellular material orculture medium when produced by recombinant DNA techniques, or chemicalprecursors, or other chemicals when chemically synthesized are includedin this invention.

In a preferred embodiment, the invention provides an isolated nucleicacid sequence comprising or consisting of:

(a) a nucleic acid sequence as shown in SEQ. ID. NOS.: 17-20, 25-28,33-36, 41-44, 49-52, 57-60, 65-68, 73-76, 81-84, 89-92, 97-100 or thatwherein T can also be U or that encodes a peptide having an amino acidsequence selected from the group consisting of: SEQ. ID. NOS: 13, 14,21, 22, 29, 30, 37, 38, 45, 46, 53, 54, 61, 62, 69, 70, 77, 78, 85, 86,93, 94, 101, 103 or that further has an amidation signal sequence(preferably GKR or GRR), at the carboxy terminus of said peptides, suchas 15, 16, 23, 24, 31, 32, 39, 40, 47, 48, 55, 56, 63, 64, 71, 72, 79,80, 87, 88, 95, 96;

(b) a nucleic acid sequence that is complimentary to a nucleic acidsequence of (a);

(c) a nucleic acid sequence that has substantial sequence homology to anucleic acid sequence of (a) or (b);

(d) a nucleic acid sequence that is an analog of a nucleic acid sequenceof (a), (b) or (c); or

(e) a nucleic acid sequence that hybridizes to a nucleic acid sequenceof (a), (b), (c) or (d) under stringent hybridization conditions.

(f) a nucleic acid sequence of (a)-(e) where T is U.

The term “sequence that has substantial sequence homology” means thosenucleic acid sequences which have slight or inconsequential sequencevariations from the sequences in (a) or (b), i.e., the sequencesfunction in substantially the same manner. The variations may beattributable to local mutations or structural modifications. Nucleicacid sequences having substantial homology include nucleic acidsequences having at least 65%, more preferably at least 85%, and mostpreferably 90-95% identity with the nucleic acid sequences as listed in(a) above. The term “sequence that hybridizes” means a nucleic acidsequence that can hybridize to a sequence of (a), (b), (c) or (d) understringent hybridization conditions. Appropriate “stringent hybridizationconditions” which promote DNA hybridization are known to those skilledin the art, or may be found in Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the followingmay be employed: 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C.; 0.2×SSC at 50° C. to 65°C.; or 2.0×SSC at 44° C. to 50° C. The stringency may be selected basedon the conditions used in the wash step. For example, the saltconcentration in the wash step can be selected from a high stringency ofabout 0.2×SSC at 50° C. In addition, the temperature in the wash stepcan be at high stringency conditions, at about 65° C.

The term “nucleic acid” is intended to include DNA and RNA and can beeither double stranded or single stranded.

The term “a nucleic acid sequence which is an analog” means a nucleicacid sequence which has been modified as compared to the sequence of(a), (b) or (c) wherein the modification does not alter the utility ofthe sequence as described herein. The modified sequence or analog mayhave improved properties over the sequence shown in (a), (b) or (c). Oneexample of a modification to prepare an analog is to replace one of thenaturally occurring bases (i.e. adenine, guanine, cytosine or thymidine)of the sequence with a modified base such as such as xanthine,hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosineand 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8 aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

Another example of a modification is to include modified phosphorous oroxygen heteroatoms in the phosphate backbone, short chain alkyl orcycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages in the nucleic acid molecule listed in(a) to (e) above. For example, the nucleic acid sequences may containphosphorothioates, phosphotriesters, methyl phosphonates, andphosphorodithioates.

A further example of an analog of a nucleic acid molecule of theinvention is a peptide nucleic acid (PNA) wherein the deoxyribose (orribose) phosphate backbone in the DNA (or RNA), is replaced with apolyamide backbone which is similar to that found in peptides (P. E.Nielsen, et al Science 1991, 254, 1497). PNA analogs have been shown tobe resistant to degradation by enzymes and to have extended lives invivo and in vitro. PNAs also bind stronger to a complimentary DNAsequence due to the lack of charge repulsion between the PNA strand andthe DNA strand. Other nucleic acid analogs may contain nucleotidescontaining polymer backbones, cyclic backbones, or acyclic backbones.For example, the nucleotides may have morpholino backbone structures(U.S. Pat. No. 5,034,506). The analogs may also contain groups such asreporter groups, a group for improving the pharmacokinetic orpharmacodynamic properties of nucleic acid sequence.

Isolated and purified nucleic acid molecules having sequences whichdiffer from the nucleic acid sequence of the invention due to degeneracyin the genetic code are also within the scope of the invention. Suchnucleic acids encode functionally equivalent peptides but differ insequence from the above mentioned sequences due to degeneracy in thegenetic code.

An isolated nucleic acid molecule of the invention which consists of DNAcan be isolated by preparing a labeled nucleic acid probe based on allor part of the nucleic acid sequences of the invention and using thislabeled nucleic acid probe to screen an appropriate DNA library (e.g. acDNA or genomic DNA library). For example, a genomic library isolatedcan be used to isolate a DNA encoding a novel peptide of the inventionby screening the library with the labeled probe using standardtechniques. Nucleic acids isolated by screening of a cDNA or genomic DNAlibrary can be sequenced by standard techniques.

An isolated nucleic acid molecule of the invention which is DNA can alsobe isolated by selectively amplifying a nucleic acid encoding a novelpeptide of the invention using the polymerase chain reaction (PCR)methods and cDNA or genomic DNA. It is possible to design syntheticoligonucleotide primers from the nucleic acid sequence of the inventionfor use in PCR. A nucleic acid can be amplified from cDNA or genomic DNAusing these oligonucleotide primers and standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis. It willbe appreciated that cDNA may be prepared from mRNA, by isolating totalcellular mRNA by a variety of techniques, for example, by using theguanidinium-thiocyanate extraction procedure of Chirgwin et al.,Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from themRNA using reverse transcriptase (for example, Moloney MLV reversetranscriptase available from Invitrogen, Carlsbad, Calif., or AMVreverse transcriptase available from Seikagaku America, Inc., St.Petersburg, Fla.).

An isolated nucleic acid molecule of the invention which is RNA can beisolated by cloning a cDNA encoding a novel peptide of the inventioninto an appropriate vector which allows for transcription of the cDNA toproduce an RNA molecule which encodes a protein of the invention. Forexample, a cDNA can be cloned downstream of a bacteriophage promoter,(e.g., a T7 promoter) in a vector, cDNA can be transcribed in vitro withT7 polymerase, and the resultant RNA can be isolated by standardtechniques.

A nucleic acid molecule of the invention may also be chemicallysynthesized using standard techniques. Various methods of chemicallysynthesizing polydeoxynucleotides are known, including solid-phasesynthesis which, like peptide synthesis, has been fully automated incommercially available DNA synthesizers (See e.g., Itakura et al. U.S.Pat. No. 4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; andItakura U.S. Pat. Nos. 4,401,796 and 4,373,071).

Determination of whether a particular nucleic acid molecule encodes anovel peptide of the invention may be accomplished by expressing thecDNA in an appropriate host cell by standard techniques, and testing theactivity of the peptide using the methods as described herein. A cDNAhaving the activity of a novel peptide of the invention so isolated canbe sequenced by standard techniques, such as dideoxynucleotide chaintermination or Maxam-Gilbert chemical sequencing, to determine thenucleic acid sequence and the predicted amino acid sequence of theencoded peptide.

The initiation codon and untranslated sequences of nucleic acidmolecules of the invention may be determined using currently availablecomputer software designed for the purpose, such as PC/Gene(IntelliGenetics Inc., Calif.). Regulatory elements can be identifiedusing conventional techniques. The function of the elements can beconfirmed by using these elements to express a reporter gene which isoperatively linked to the elements. These constructs may be introducedinto cultured cells using standard procedures. In addition toidentifying regulatory elements in DNA, such constructs may also be usedto identify proteins interacting with the elements, using techniquesknown in the art.

The sequence of a nucleic acid molecule of the invention may be invertedrelative to its normal presentation for transcription to produce anantisense nucleic acid molecule which are more fully described herein.In particular, the nucleic acid sequences contained in the nucleic acidmolecules of the invention or a fragment thereof, may be invertedrelative to its normal presentation for transcription to produceantisense nucleic acid molecules.

The invention also provides nucleic acids encoding fusion proteinscomprising a novel protein of the invention and a selected protein, or aselectable marker protein (see below).

Also provided are portions of the nucleic acid sequence encodingfragments, functional domains or antigenic determinants of the TCAPpeptide. The present invention also provides for the use of portions ofthe sequence as probes and PCR primers for TCAP as well as fordetermining functional aspects of the sequence.

One of ordinary skill in the art is now enabled to identify and isolateTCAP encoding nucleic acids or cDNAs that are allelic variants of thedisclosed sequences, using standard hybridization screening or PCRtechniques.

II. Novel Proteins of the Invention

The invention further broadly contemplates an isolated TCAP peptide. Theterm “TCAP peptide” as used herein includes all homologs, analogs,fragments or derivatives of the TCAP peptide.

The term “analog” in reference to peptides includes any peptide havingan amino acid residue sequence substantially identical to the human ormouse TCAP sequence specifically shown herein in which one or moreresidues have been conservatively substituted with a functionallysimilar residue and which displays the ability to mimic TCAP asdescribed herein. Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue such as alanine,isoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another. The phrase“conservative substitution” also includes the use of a chemicallyderivatized residue in place of a non-derivatized residue provided thatsuch polypeptide displays the requisite activity.

The term “derivative” reference to peptides refers to a peptide havingone or more residues chemically derivatized by reaction of a functionalside group. Such derivatized molecules include for example, thosemolecules in which free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as derivatives are those peptides which contain one ormore naturally occurring amino acid derivatives of the twenty standardamino acids. For examples: 4-hydroxyproline may be substituted forproline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions or residues relative tothe sequence of a polypeptide whose sequence is shown herein, so long asthe requisite activity is maintained.

In one embodiment, the isolated TCAP peptide consists of 38-41 aminoacid residues of the carboxy terminus of a teneurin-like protein with orwithout an amidation signal at the carboxy terminus. In one embodiment,the amidation signal consists of the amino acid sequence GKR or GRR(preTCAP). In another embodiment, the TCAP peptide comprises sequencessubstantially identity to the above-noted peptides or comprising anobvious chemical equivalent thereof. It also includes peptides sequence+/−amino acids at the amino and/or carboxy terminus of the above-notedTCAP peptide sequences. In yet another embodiment, the inventionincludes fusion proteins, comprising the TCAP peptide, labeled TCAPpeptides, analogs, homologs and variants thereof.

In one embodiment, the TCAP peptide is a rainbow trout, zebrafish,human, mouse, G. gallus or D. melanogaster TCAP. In another embodiment,the TCAP peptides have the sequence selected from the group consistingof: SEQ. ID. NOS: 13, 14, 21, 22, 29, 30, 37, 38, 45, 46, 53, 54, 61,62, 69, 70, 77, 78, 85, 86, 93, 94, 101, 103 or that further has anamidation signal sequence (preferably GKR or GRR), at the carboxyterminus of said peptides, such as 15, 16, 23, 24, 31, 32, 39, 40, 47,48, 55, 56, 63, 64, 71, 72, 79, 80, 87, 88, 95, 96;

In another embodiment, the peptide of the invention is a TCAP-3 and hasthe following amino acid sequence motif:

SEQ. ID. NO: 135 QLLSXaa₁Xaa₂KVXaa₃GYDGYYVLSXaa₄EQYPELADSANNXaa₅QFLRQSEI

Where Xaa_(i) is G, S, or A; Xaa₂ is G or R; Xaa₃ is L or Q; Xaa₄ andXaa₅ are independently V or I. In one embodiment, the TCAP-3 is a humanor mouse TCAP-3. In another embodiment, the TCAP-3 has SEQ. ID. NO: 13,21, 53 or 85.

Within the context of the present invention, a peptide of the inventionmay include various structural forms of the primary peptide which retainbiological activity. For example, a peptide of the invention may be inthe form of acidic or basic salts or in neutral form. In addition,individual amino acid residues may be modified by oxidation orreduction.

In addition to the full-length amino acid sequence, the peptide of thepresent invention may also include truncations, analogs and homologs ofthe peptide and truncations thereof as described herein. Truncatedpeptides or fragments may comprise peptides of at least 5, preferably 10and more preferably 15 amino acid residues of the sequence listed above.

The invention further provides polypeptides comprising at least onefunctional domain or at least one antigenic determinant of a TCAPpeptide.

Analogs of the protein of the invention and/or truncations thereof asdescribed herein, may include, but are not limited to an amino acidsequence containing one or more amino acid substitutions, insertions,deletions and/or mutations. Amino acid substitutions may be of aconserved or non-conserved nature. Conserved amino acid substitutionsinvolve replacing one or more amino acids of the peptides of theinvention with amino acids of similar charge, size, and/orhydrophobicity characteristics. When only conserved substitutions aremade the resulting analog should be functionally equivalent.Non-conserved substitutions involve replacing one or more amino acids ofthe amino acid sequence with one or more amino acids which possessdissimilar charge, size, and/or hydrophobicity characteristics.

One or more amino acid insertions may be introduced into the amino acidsequences of the invention. Amino acid insertions may consist of singleamino acid residues or sequential amino acids ranging from 2 to 15 aminoacids in length. For example, amino acid insertions may be used todestroy target sequences so that the peptide is no longer active. Thisprocedure may be used in vivo to inhibit the activity of the peptide ofthe invention.

Deletions may consist of the removal of one or more amino acids, ordiscrete portions from the amino acid sequence of the TCAP peptide. Thedeleted amino acids may or may not be contiguous.

Analogs of a protein of the invention may be prepared by introducingmutations in the nucleotide sequence encoding the peptide. Mutations maybe introduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes an analog having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures may be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Deletion or truncation of a peptide of the invention may alsobe constructed by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al (MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor LaboratoryPress, 1989).

The peptides of the invention also include homologs of the amino acidsequence of the TCAP peptide, mutated TCAP peptides and/or truncationsthereof as described herein. Such homologs are proteins whose amino acidsequences are comprised of amino acid sequences that hybridize understringent hybridization conditions (see discussion of stringenthybridization conditions herein) with a probe used to obtain a peptideof the invention. Homologs of a peptide of the invention will have thesame regions which are characteristic of the protein.

A homologous peptide includes a peptide with an amino acid sequencehaving at least 70%, preferably 80-95% identity with the amino acidsequence of the TCAP peptide.

The invention also contemplates isoforms of the peptides of theinvention. An isoform contains the same number and kinds of amino acidsas a peptide of the invention, but the isoform has a different molecularstructure. The isoforms contemplated by the present invention are thosehaving the same properties as a peptide of the invention as describedherein.

The proteins of the invention (including e.g., truncations, analogs,etc.) may be prepared using recombinant DNA methods. Accordingly,nucleic acid molecules of the present invention having a sequence thatencodes a peptide of the invention may be incorporated according toprocedures known in the art into an appropriate expression vector thatensures good expression of the peptide. Possible expression vectorsinclude but are not limited to cosmids, plasmids, or modified viruses(e.g., replication defective retroviruses, adenoviruses andadeno-associated viruses), so long as the vector is compatible with thehost cell used. The expression “vectors suitable for transformation of ahost cell”, means that the expression vectors contain a nucleic acidmolecule of the invention and regulatory sequences, selected on thebasis of the host cells to be used for expression, which are operativelylinked to the nucleic acid molecule. “Operatively linked” is intended tomean that the nucleic acid is linked to regulatory sequences in a mannerthat allows expression of the nucleic acid.

The invention therefore contemplates a recombinant expression vector ofthe invention containing a nucleic acid molecule of the invention, or afragment thereof, and the necessary regulatory sequences for thetranscription and translation of the inserted peptide-sequence. Suitableregulatory sequences may be derived from a variety of sources, includingbacterial, fungal, or viral genes (For example, see the regulatorysequences described in Goeddel, Gene Expression Technology Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Selection ofappropriate regulatory sequences is dependent on the host cell chosen,and may be readily accomplished by one of ordinary skill in the art.Examples of such regulatory sequences include: a transcriptionalpromoter and enhancer or RNA polymerase binding sequence, a ribosomalbinding sequence, including a translation initiation signal.Additionally, depending on the host cell chosen and the vector employed,other sequences, such as an origin of replication, additional DNArestriction sites, enhancers, and sequences conferring inducibility oftranscription may be incorporated into the expression vector. It willalso be appreciated that the necessary regulatory sequences may besupplied by the native peptide and/or its flanking regions.

The invention further provides a recombinant expression vectorcomprising a DNA nucleic acid molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner that allowsfor expression, by transcription of the DNA molecule, of an RNA moleculewhich is antisense to a nucleotide sequence of the invention. Regulatorysequences operatively linked to the antisense nucleic acid can be chosenwhich direct the continuous expression of the antisense RNA molecule.

The recombinant expression vectors of the invention may also contain aselectable marker gene that facilitates the selection of host cellstransformed or transfected with a recombinant molecule of the invention.Examples of selectable marker genes are genes encoding a protein such asG418 and hygromycin which confer resistance to certain drugs,β-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. Transcription of the selectable marker gene is monitored bychanges in the concentration of the selectable marker protein such asβ-galactosidase, chloramphenicol acetyltransferase, or fireflyluciferase. If the selectable marker gene encodes a protein conferringantibiotic resistance such as neomycin resistance transformant cells canbe selected with G418. Cells that have incorporated the selectablemarker gene will survive, while the other cells die. This makes itpossible to visualize and assay for expression of recombinant expressionvectors of the invention and in particular to determine the effect of amutation on expression and phenotype. It will be appreciated thatselectable markers can be introduced on a separate vector from thenucleic acid of interest.

Recombinant expression vectors can be introduced into host cells toproduce a transformed host cell. Accordingly, the invention includes ahost cell comprising a recombinant expression vector of the invention.The term “transformed host cell” is intended to include prokaryotic andeukaryotic cells which have been transformed or transfected with arecombinant expression vector of the invention. The terms “transformedwith”, “transfected with”, “transformation” and “transfection” areintended to encompass introduction of nucleic acid (e.g. a vector) intoa cell by one of many possible techniques known in the art. Prokaryoticcells can be transformed with nucleic acid by, for example,electroporation or calcium-chloride mediated transformation. Nucleicacid can be introduced into mammalian cells via conventional techniquessuch as calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofectin, electroporation ormicroinjection. Suitable methods for transforming and transfecting hostcells can be found in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), andother such laboratory textbooks.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the peptides of the invention may be expressedin bacterial cells such as E. coli, Pseudomonas, Bacillus subtillus,insect cells (using baculovirus), yeast cells or mammalian cells. Othersuitable host cells can be found in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1991).

As an example, to produce TCAP peptides recombinantly, for example, E.coli can be used using the T7 RNA polymerase/promoter system using twoplasmids or by labeling of plasmid-encoded proteins, or by expression byinfection with M13 Phage mGPI-2. E. coli vectors can also be used withPhage lamba regulatory sequences, by fusion protein vectors (e.g. IacZand trpE), by maltose-binding protein fusions, and byglutathione-S-transferase fusion proteins.

Alternatively, a TCAP peptide can be expressed in insect cells usingbaculoviral vectors, or in mammalian cells using vaccinia virus. Forexpression in mammalian cells, the cDNA sequence may be ligated toheterologous promoters and introduced into cells, such as COS cells toachieve transient or long-term expression. The stable integration of thechimeric gene construct may be maintained in mammalian cells bybiochemical selection, such as neomycin and mycophoenolic acid.

The TCAP DNA sequence can be altered using procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence alterationwith the use of specific oligonucleotides together with PCR.

The cDNA sequence or portions thereof, or a mini gene consisting of acDNA with an intron and its own promoter, is introduced into eukaryoticexpression vectors by conventional techniques. These vectors permit thetranscription of the cDNA in eukaryotic cells by providing regulatorysequences that initiate and enhance the transcription of the cDNA andensure its proper splicing and polyadenylation. The endogenous TCAP genepromoter can also be used. Different promoters within vectors havedifferent activities which alters the level of expression of the cDNA.In addition, certain promoters can also modulate function such as theglucocorticoid-responsive promoter from the mouse mammary tumor virus.

Some of the vectors listed contain selectable markers or neo bacterialgenes that permit isolation of cells by chemical selection. Stablelong-term vectors can be maintained in cells as episomal, freelyreplicating entities by using regulatory elements of viruses. Cell linescan also be produced which have integrated the vector into the genomicDNA. In this manner, the gene product is produced on a continuous basis.

Vectors are introduced into recipient cells by various methods includingcalcium phosphate, strontium phosphate, electroporation, lipofection,DEAE dextran, microinjection, or by protoplast fusion. Alternatively,the cDNA can be introduced by infection using viral vectors.

TCAP peptides may also be isolated from cells or tissues, includingmammalian cells or tissues, in which the peptide is normally expressed.

The protein may be purified by conventional purification methods knownto those in the art, such as chromatography methods, high performanceliquid chromatography methods or precipitation.

For example, an anti-TCAP antibody (as described below) may be used toisolate a TCAP peptide, which is then purified by standard methods.

The peptides of the invention may also be prepared by chemical synthesisusing techniques well known in the chemistry of proteins such as solidphase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) orsynthesis in homogenous solution (Houbenweyl, 1987, Methods of OrganicChemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).

III. Uses

The present invention includes all uses of the nucleic acid molecules,TCAP peptides and preTCAP peptides of the invention including, but notlimited to, the preparation of antibodies and antisenseoligonucleotides, the preparation of experimental systems to study TCAP,the isolation of substances that can bind or modulate TCAP expressionand/or activity as well as the use of the TCAP nucleic acid sequencesand peptides and modulators thereof in diagnostic and therapeuticapplications. Some of the uses are further described below.

(a) Antibodies

The isolation of the TCAP peptide enables the preparation of antibodiesspecific for TCAP. Accordingly, the present invention provides anantibody that binds to a TCAP peptide and/or a protein containing a TCAPpeptide, such as preTCAP.

Conventional methods can be used to prepare the antibodies. For example,by using a TCAP, polyclonal antisera or monoclonal antibodies can bemade using standard methods. A mammal, (e.g., a mouse, hamster, orrabbit) can be immunized with an immunogenic form of the peptide whichelicits an antibody response in the mammal. Techniques for conferringimmunogenicity on a peptide include conjugation to carriers or othertechniques well known in the art. For example, the protein or peptidecan be administered in the presence of adjuvant. The progress ofimmunization can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassay procedures can be usedwith the immunogen as antigen to assess the levels of antibodies.Following immunization, antisera can be obtained and, if desired,polyclonal antibodies isolated from the sera.

To produce monoclonal antibodies, antibody-producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art, (e.g., the hybridoma technique originally developed by Kohlerand Milstein (Nature 256, 495-497 (1975)) as well as other techniquessuch as the human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4, 72 (1983)), the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al. Monoclonal Antibodies in CancerTherapy (1985) Allen R. Bliss, Inc., pages 77-96), and screening ofcombinatorial antibody libraries (Huse et al., Science 246, 1275(1989)). Hybridoma cells can be screened immunochemically for productionof antibodies specifically reactive with the peptide and the monoclonalantibodies can be isolated. Therefore, the invention also contemplateshybridoma cells secreting monoclonal antibodies with specificity forTCAP.

The term “antibody” as used herein is intended to include fragmentsthereof which also specifically react with TCAP. Antibodies can befragmented using conventional techniques and the fragments screened forutility in the same manner as described above. For example, F(ab′)2fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)2 fragment can be further treated to produce Fab′fragments.

Chimeric antibody derivatives, i.e., antibody molecules that combine anon-human animal variable region and a human constant region are alsocontemplated within the scope of the invention. Chimeric antibodymolecules can include, for example, the antigen binding domain from anantibody of a mouse, rat, or other species, with human constant regions.Conventional methods may be used to make chimeric antibodies containingthe immunoglobulin variable region which recognizes the gene product ofTCAP antigen of the invention (See, for example, Morrison et al., Proc.Natl Acad. Sci. U.S.A. 81, 6851 (1985); Takeda et al., Nature 314, 452(1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat.No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496;European Patent Publication 0173494, United Kingdom patent GB 2177096B).It is expected that chimeric antibodies would be less immunogenic in ahuman subject than the corresponding non-chimeric antibody.

Monoclonal or chimeric antibodies specifically reactive with a peptideof the invention as described herein can be further humanized byproducing human constant region chimeras, in which parts of the variableregions, particularly the conserved framework regions of theantigen-binding domain, are of human origin and only the hypervariableregions are of non-human origin. Such immunoglobulin molecules may bemade by techniques known in the art, (e.g., Teng et al., Proc. Natl.Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., ImmunologyToday, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)),and PCT Publication WO92/06193 or EP 0239400). Humanized antibodies canalso be commercially produced (Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.)

Specific antibodies, or antibody fragments, reactive against TCAPpeptide may also be generated by screening expression libraries encodingimmunoglobulin genes, or portions thereof, expressed in bacteria withpeptides produced from the nucleic acid molecules encoding TCAP. Forexample, complete Fab fragments, VH regions and FV regions can beexpressed in bacteria using phage expression libraries (See for exampleWard et al., Nature 341, 544-546: (1989); Huse et al., Science 246,1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).Alternatively, a SCID-hu mouse, for example the model developed byGenpharm, can be used to produce antibodies or fragments thereof.

(b) Antisense Oligonucleotides

Isolation of a nucleic acid molecule encoding TCAP enables theproduction of antisense oligonucleotides that can modulate theexpression and/or activity of TCAP. Accordingly, the present inventionprovides an antisense oligonucleotide that is complimentary to a nucleicacid sequence encoding TCAP.

The term “antisense oligonucleotide” as used herein means a nucleotidesequence that is complimentary to its target.

The term “oligonucleotide” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars, and intersugar (backbone) linkages. The term alsoincludes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Suchmodified or substituted oligonucleotides may be preferred over naturallyoccurring forms because of properties such as enhanced cellular uptake,or increased stability in the presence of nucleases. The term alsoincludes chimeric oligonucleotides which contain two or more chemicallydistinct regions. For example, chimeric oligonucleotides may contain atleast one region of modified nucleotides that confer beneficialproperties (e.g. increased nuclease resistance, increased uptake intocells), or two or more oligonucleotides of the invention may be joinedto form a chimeric oligonucleotide.

The antisense oligonucleotides of the present invention may beribonucleic or deoxyribonucleic acids and may contain naturallyoccurring bases including adenine, guanine, cytosine, thymidine anduracil. The oligonucleotides may also contain modified bases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and otheralkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-azacytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, thymidines,cytosines, adenines, or guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine.

Other antisense oligonucleotides of the invention may contain modifiedphosphorous, oxygen heteroatoms in the phosphate backbone, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. For example, the antisenseoligonucleotides may contain phosphorothioates, phosphotriesters, methylphosphonates, and phosphorodithioates. In an embodiment of the inventionthere are phosphorothioate bonds links between the four to six3′-terminus bases. In another embodiment phosphorothioate bonds link allthe nucleotides.

The antisense oligonucleotides of the invention may also comprisenucleotide analogs that may be better suited as therapeutic orexperimental reagents. An example of an oligonucleotide analogue is apeptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphatebackbone in the DNA (or RNA), is replaced with a polyamide backbonewhich is similar to that found in peptides (P. E. Nielsen, et al Science1991, 254, 1497). PNA analogues have been shown to be resistant todegradation by enzymes and to have extended lives in vivo and in vitro.PNAs also bind stronger to a complimentary DNA sequence due to the lackof charge repulsion between the PNA strand and the DNA strand. Otheroligonucleotides may contain nucleotides containing polymer backbones,cyclic backbones, or acyclic backbones. For example, the nucleotides mayhave morpholino backbone structures (U.S. Pat. No. 5,034,506).Oligonucleotides may also contain groups such as reporter groups, agroup for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an antisense oligonucleotide. Antisense oligonucleotides may alsohave sugar mimetics.

The antisense nucleic acid molecules may be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. The antisense nucleic acid molecules of the invention or a fragmentthereof, may be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed with mRNA or the native gene e.g.phosphorothioate derivatives and acridine substituted nucleotides. Theantisense sequences may be produced biologically using an expressionvector introduced into cells in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense sequences are producedunder the control of a high efficiency regulatory region, the activityof which may be determined by the cell type into which the vector isintroduced.

The antisense oligonucleotides may be introduced into tissues or cellsusing techniques in the art including vectors (retroviral vectors,adenoviral vectors and DNA virus vectors) or physical techniques such asmicroinjection. The antisense oligonucleotides may be directlyadministered in vivo or may be used to transfect cells in vitro whichare then administered in vivo. In one embodiment, the antisenseoligonucleotide may be delivered to macrophages and/or endothelial cellsin a liposome formulation.

(c) Diagnostic Assays

The findings by the present inventors that TCAP is involved ininhibiting neuronal cell proliferation, in inducing an anxiogenicresponse and in inhibiting cell death in cells subject to stress allowsdevelopment of diagnostic assays, particularly for conditions associatedwith the aberrant regulation of neuronal growth.

Accordingly, the present invention provides a method of detecting acondition associated with TCAP or preTCAP expression comprising assayinga sample for (a) a nucleic acid molecule encoding a TCAP peptide or afragment thereof or (b) a TCAP protein or a fragment thereof. The TCAPpeptide preferably has a sequence as shown in SEQ. ID. NOS.: 13, 14, 21,22, 29, 30, 37, 38, 45, 46, 53, 54, 61, 62, 69, 70, 77, 78, 85, 86, 93,94, 101, 103. In one particular embodiment of the invention thecondition is associated with the aberrant regulation of neuronal growth.Neuronal growth may include somatic and process development, mitogenesisor migration. Aberrant regulation of neuronal growth may occur via adisturbance in interneuronal connections and the associated signalmolecules. Examples of such conditions include learning deficits, mentalretardation, autism, schizophrenia, Alzheimer's Disease, Parkinson'sDisease as well as affective disorders such as panic disorder,depression, anorexia nervosa and obsessive-compulsive disorder.

(i) Nucleic Acid Molecules

The nucleic acid molecules encoding TCAP as described herein orfragments thereof, allow those skilled in the art to constructnucleotide probes for use in the detection of nucleotide sequencesencoding TCAP or fragments thereof in samples, preferably biologicalsamples such as cells, tissues and bodily fluids. The probes can beuseful in detecting the presence of a condition associated with TCAPexpression or monitoring the progress of such a condition. Accordingly,the present invention provides a method for detecting a nucleic acidmolecule encoding a TCAP comprising contacting the sample with anucleotide probe capable of hybridizing with the nucleic acid moleculeto form a hybridization product, under conditions which permit theformation of the hybridization product, preferably under stringentconditions, and assaying for the hybridization product.

Example of probes that may be used in the above method include fragmentsof the nucleic acid sequences shown in SEQ. ID. NOS.:18-20, 25-28,33-36, 41-44, 49-52, 57-60, 65-68, 73-76, 81-84, 89-92, 97-100 or thatwherein T can also be U or that encodes a peptide having an amino acidsequence selected from the group consisting of: SEQ. ID. NOS: 13, 14,21, 22, 29, 30, 37, 38, 45, 46, 53, 54, 61, 62, 69, 70, 77, 78, 85, 86,93, 94, 101, 103 or that further has an amidation signal sequence(preferably GKR or GRR), at the carboxy terminus of said peptides, suchas 15, 16, 23, 24, 31, 32, 39, 40, 47, 48, 55, 56, 63, 64, 71, 72, 79,80, 87, 88, 95, 96. A nucleotide probe may be labelled with a detectablesubstance such as a radioactive label which provides for an adequatesignal and has sufficient half-life such as 32P, 3H, 14C or the like.Other detectable substances which may be used include antigens that arerecognized by a specific labelled antibody, fluorescent compounds,enzymes, antibodies specific for a labelled antigen, andchemiluminescence. An appropriate label may be selected having regard tothe rate of hybridization and binding of the probe to the nucleic acidto be detected and the amount of nucleic acid available forhybridization. Labelled probes may be hybridized to nucleic acids onsolid supports such as nitrocellulose filters or nylon membranes asgenerally described in Sambrook et al, 1989, Molecular Cloning, ALaboratory Manual (2nd ed.). The nucleotide probes may be used to detectgenes, preferably in human cells, that hybridize to the nucleic acidmolecule of the present invention preferably, nucleic acid moleculeswhich hybridize to the nucleic acid molecule of the invention understringent hybridization conditions as described herein.

Nucleic acid molecules encoding a TCAP peptide can be selectivelyamplified in a sample using the polymerase chain reaction (PCR) methodsand cDNA or genomic DNA. It is possible to design syntheticoligonucleotide primers from the nucleotide sequence shown in FIGS. 1-5for use in PCR. A nucleic acid can be amplified from cDNA or genomic DNAusing oligonucleotide primers and standard PCR amplification techniques.The amplified nucleic acid can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. cDNA may be prepared from mRNA,by isolating total cellular mRNA by a variety of techniques, forexample, by using the guanidinium-thiocyanate extraction procedure ofChirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNA is thensynthesized from the mRNA using reverse transcriptase (for example,Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda,Md., or AMV reverse transcriptase available from Seikagaku America,Inc., St. Petersburg, Fla.).

Patients may be screened routinely using probes to detect the presenceof a TCAP gene by a variety of techniques. Genomic DNA used for thediagnosis may be obtained from body cells, such as those present in theblood, tissue biopsy, surgical specimen, or autopsy material. The DNAmay be isolated and used directly for detection of a specific sequenceor may be PCR amplified prior to analysis. RNA or cDNA may also be used.To detect a specific DNA sequence hybridization using specificoligonucleotides, direct DNA sequencing, restriction enzyme digest,RNase protection, chemical cleavage, and ligase-mediated detection areall methods which can be utilized. Oligonucleotides specific to mutantsequences can be chemically synthesized and labelled radioactively withisotopes, or non-radioactively using biotin tags, and hybridized toindividual DNA samples immobilized on membranes or other solid-supportsby dot-blot or transfer from gels after electrophoresis. The presence orabsence of these mutant sequences is then visualized using methods suchas autoradiography, fluorometry, or colorimetric reaction. Suitable PCRprimers can be generated which are useful for example in amplifyingportions of the subject sequence containing identified mutations. Othernucleotide sequence amplification techniques may be used, such asligation-mediated PCR, anchored PCR and enzymatic amplification as wouldbe understood by those skilled in the art.

Sequence alterations may also generate fortuitous restriction enzymerecognition sites that are revealed by the use of appropriate enzymedigestion followed by gel-blot hybridization. DNA fragments carrying thesite (normal or mutant) are detected by their increase or reduction insize, or by the increase or decrease of corresponding restrictionfragment numbers. Genomic DNA samples may also be amplified by PCR priorto treatment with the appropriate restriction enzyme and the fragmentsof different sizes are visualized under UV light in the presence ofethidium bromide after gel electrophoresis.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels. Small sequence deletions and insertions can be visualized byhigh-resolution gel electrophoresis. Small deletions may also bedetected as changes in the migration pattern of DNA heteroduplexes innon-denaturing gel electrophoresis. Alternatively, a single basesubstitution mutation may be detected based on differential primerlength in PCR. The PCR products of the normal and mutant gene could bedifferentially detected in acrylamide gels.

Nuclease protection assays (S1 or ligase-mediated) also reveal sequencechanges at specific locations. Alternatively, to confirm or detect apolymorphism restriction mapping changes ligated PCR, ASO, REF-SSCP andSSCP may be used. Both REF-SSCP and SSCP are mobility shift assays thatare based upon the change in conformation due to mutations.

DNA fragments may also be visualized by methods in which the individualDNA samples are not immobilized on membranes. The probe and targetsequences may be in solution or the probe sequence may be immobilized.Autoradiography, radioactive decay, spectrophotometry, and fluorometrymay also be used to identify specific individual genotypes.

(ii) Proteins

The TCAP protein may be detected in a sample using antibodies that bindto the protein as described in detail above. Accordingly, the presentinvention provides a method for detecting a TCAP protein comprisingcontacting the sample with an antibody that binds to TCAP and which iscapable of being detected after it becomes bound to the TCAP in thesample.

Antibodies specifically reactive with TCAP, or derivatives thereof, suchas enzyme conjugates or labeled derivatives, may be used to detect TCAPin various biological materials, for example they may be used in anyknown immunoassays which rely on the binding interaction between anantigenic determinant of TCAP, and the antibodies. Examples of suchassays are radioimmunoassays, enzyme immunoassays (e.g. ELISA),immunofluorescence, immunoprecipitation, latex agglutination,hemagglutination and histochemical tests. Thus, the antibodies may beused to detect and quantify mutated TCAP in a sample in order todetermine its role in particular cellular events or pathological states,and to diagnose and treat such pathological states.

In particular, the antibodies of the invention may be used inimmuno-histochemical analyses, for example, at the cellular andsub-subcellular level, to detect TCAP, to localize it to particularcells and tissues and to specific subcellular locations, and toquantitate the level of expression.

Cytochemical techniques known in the art for localizing antigens usinglight and electron microscopy may be used to detect TCAP. Generally, anantibody of the invention may be labelled with a detectable substanceand TCAP may be localised in tissue based upon the presence of thedetectable substance. Examples of detectable substances include variousenzymes, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,biotin, alkaline phosphatase, β-galactosidase, or acetylcholinesterase;examples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include radioactive iodine I-125, I-131 or3-H. Antibodies may also be coupled to electron dense substances, suchas ferritin or colloidal gold, which are readily visualized by electronmicroscopy.

Indirect methods may also be employed in which the primaryantigen-antibody reaction is amplified by the introduction of a secondantibody, having specificity for the antibody reactive against TCAP. Byway of example, if the antibody having specificity against TCAP is arabbit IgG antibody, the second antibody may be goat anti-rabbitgamma-globulin labelled with a detectable substance as described herein.

Where a radioactive label is used as a detectable substance, TCAP may belocalized by autoradiography. The results of autoradiography may bequantitated by determining the density of particles in theautoradiographs by various optical methods, or by counting the grains.

(d) Experimental Systems

Eukaryotic expression systems are preferred and can be used for manystudies of TCAP encoding genes and gene product(s) including theproduction of large amounts of the peptide for isolation andpurification, to use cells expressing the TCAP peptide as a functionalassay system for antibodies generated against the peptide or to testeffectiveness of pharmacological agents, to study the function of thenormal complete peptide, specific portions of the peptide, or ofnaturally occurring and artificially produced mutant peptides.

Using the techniques mentioned, the expression vectors containing theTCAP peptide cDNA sequence or portions thereof can be introduced into avariety of mammalian cells from other species or into non-mammaliancells.

The recombinant cloning vector, according to this invention, comprisesthe selected DNA of the DNA sequences of this invention for expressionin a suitable host. The DNA is operatively linked in the vector to anexpression control sequence in the recombinant DNA molecule so that TCAPpeptide protein can be expressed. The expression control sequence may beselected from the group consisting of sequences that control theexpression of genes of eukaryotic cells and their viruses andcombinations thereof. The expression control sequence may be selectedfrom the group consisting of the lac system, the trp system, the tacsystem, the trc system, major operator and promoter regions of phagelambda, the control region of the fd coat protein, early and latepromoters of TCAP, promoters derived from polyoma, adenovirus,retrovirus, baculovirus, simian virus, 3-phosphoglycerate kinasepromoter, yeast acid phosphatase promoters, yeast alpha-mating factorsand combinations thereof.

Expression of the TCAP peptide in heterologous cell systems may also beused to demonstrate structure-function relationships as well as toprovide cell lines for the purposes of drug screening. Inserting a TCAPDNA sequence into a plasmid expression vector to transfect cells is auseful method to test the influence of the peptide on various cellularbiochemical parameters including the identification of substrates aswell as activators and inhibitors of the gene. Plasmid expressionvectors containing either the entire coding sequence for TCAP, or forportions thereof, can be used in in vitro mutagenesis experiments thatwill identify portions of the protein crucial for function. The DNAsequence can be manipulated in studies to understand the expression ofthe gene and its product. The changes in the sequence may or may notalter the expression pattern in terms of relative quantities,tissue-specificity and functional properties.

The invention also provides methods for examining the function of theTCAP peptide encoded by the nucleic acid molecules of the invention.Cells, tissues, and non-human animals lacking in expression or partiallylacking in expression of the peptide may be developed using recombinantmolecules of the invention having specific deletion or insertionmutations in the nucleic acid molecule of the invention. A recombinantmolecule may be used to inactivate or alter the endogenous gene byhomologous recombination, and thereby create a deficient cell, tissue oranimal. Such a mutant cell, tissue or animal may be used to definespecific cell populations, developmental patterns and in vivo processes,normally dependent on the protein encoded by the nucleic acid moleculeof the invention.

Immortalized TCAP responsive cell lines can also be used to identifymodulators of TCAP such as noted in Example 13. It can also be used toidentify effect of TCAP and TCAP modulators on particular markers. In sofar as these markers are associated with the regulation of a medicalcondition, TCAP and/or the TCAP modulators may be used in the diagnosis,regulation, and/or treatment of said medical condition.

(e) TCAP Modulators

In addition to antibodies and antisense oligonucleotides describedabove, other substances that modulate TCAP expression or activity mayalso be identified.

(i) Substances that Bind/Modulate TCAP

Substances that affect TCAP activity can be identified based on theirability to bind to TCAP.

Substances which can bind with the TCAP of the invention may beidentified by reacting the TCAP with a substance which potentially bindsto TCAP, and assaying for complexes, for free substance, or fornon-complexed TCAP, or for activation of TCAP. In particular, a yeasttwo hybrid assay system may be used to identify proteins which interactwith TCAP (Fields, S, and Song, O., 1989, Nature, 340:245-247). Systemsof analysis which also may be used include ELISA.

Accordingly, the invention provides a method of identifying substanceswhich can bind with TCAP, comprising the steps of:

-   1. reacting TCAP and a test substance, under conditions which allow    for formation of a complex between the TCAP and the test substance,    and-   2. assaying for complexes of TCAP and the test substance, for free    substance or for non complexed TCAP, wherein the presence of    complexes indicates that the test substance is capable of binding    TCAP.

In another embodiment the invention provides a method of identifyingsubstances that can modulate TCAP activity, such as by binding to TCAPor a TCAP substrate and thus potentially compete (i.e. inhibit TCAPactivity), or enhance TCAP/substrate interaction (i.e enhancing TCAPactivity), the method comprising:

-   -   1. reacting TCAP and a TCAP substate and a test substance, under        conditions which allow for formation of a complex between the        TCAP and the TCAP substrate, and    -   2. assaying for complexes of TCAP and the test substance, TCAP        and TCAP substate, TCAP substrate and test substance, for free        substance or for non complexed TCAP or TCAP substrate, wherein        the presence of complexes with the test substance indicates that        the test substance is capable of binding TCAP or TCAP substrate,        as the case may be.

In another embodiment, a method of identifying modulators of TCAPcomprises the use of a cell line that has known reaction to TCAP thatcan be monitored and monitoring said reaction in the presence of TCAPand a potential modulator.

The TCAP peptide used in the assay may have the amino acid sequenceshown in SEQ. ID. NOS: 14, 21, 22, 29, 30, 37, 38, 45, 46, 53, 54, 61,62, 69, 70, 77, 78, 85, 86, 93, 94, 101, 103 or may be a fragment,analog, derivative, homolog or mimetic thereof as described herein.

Conditions which permit the formation of substance and TCAP complexesmay be selected having regard to factors such as the nature and amountsof the substance and the peptide.

The substance-peptide complex, free substance or non-complexed peptidesmay be isolated by conventional isolation techniques, for example,salting out, chromatography, electrophoresis, gel filtration,fractionation, absorption, polyacrylamide gel electrophoresis,agglutination, or combinations thereof. To facilitate the assay of thecomponents, antibody against TCAP or the substance, or labelled TCAP, ora labelled substance may be utilized. The antibodies, proteins, orsubstances may be labelled with a detectable substance as describedabove.

TCAP, or the substance used in the method of the invention may beinsolubilized. For example, TCAP or substance may be bound to a suitablecarrier. Examples of suitable carriers are agarose, cellulose, dextran,Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper,ion-exchange resin, plastic film, plastic tube, glass beads,polyamine-methyl vinyl-ether-maleic acid copolymer, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carriermay be in the shape of, for example, a tube, test plate, beads, disc,sphere etc.

The insolubilized peptide or substance may be prepared by reacting thematerial with a suitable insoluble carrier using known chemical orphysical methods, for example, cyanogen bromide coupling.

The peptide or substance may also be expressed on the surface of a cellusing the methods described herein.

The invention also contemplates assaying for an antagonist or agonist ofthe action of TCAP.

It will be understood that the agonists and antagonists that can beassayed using the methods of the invention may act on one or more of thebinding sites on the protein or substance including agonist bindingsites, competitive antagonist binding sites, non-competitive antagonistbinding sites or allosteric sites.

The invention also makes it possible to screen for antagonists thatinhibit the effects of an agonist of TCAP. Thus, the invention may beused to assay for a substance that competes for the same binding site ofTCAP.

(ii) Peptide Mimetics

The present invention also includes peptide mimetics of TCAP. “Peptidemimetics” are structures which serve as substitutes for peptides ininteractions between molecules (See Morgan et al (1989), Ann. ReportsMed. Chem. 24:243-252 for a review). Peptide mimetics include syntheticstructures which may or may not contain amino acids and/or peptide bondsbut retain the structural and functional features of a peptide, orenhancer or inhibitor of the invention. Peptide mimetics also includepeptoids, oligopeptoids (Simon et al (1972) Proc. Natl. Acad, Sci USA89:9367); and peptide libraries containing peptides of a designed lengthrepresenting all possible sequences of amino acids corresponding to apeptide of the invention.

Peptide mimetics may be designed based on information obtained bysystematic replacement of L-amino acids by D-amino acids, replacement ofside chains with groups having different electronic properties, and bysystematic replacement of peptide bonds with amide bond replacements.Local conformational constraints can also be introduced to determineconformational requirements for activity of a candidate peptide mimetic.The mimetics may include isosteric amide bonds, or D-amino acids tostabilize or promote reverse turn conformations and to help stabilizethe molecule. Cyclic amino acid analogues may be used to constrain aminoacid residues to particular conformational states. The mimetics can alsoinclude mimics of inhibitor peptide secondary structures. Thesestructures can model the 3-dimensional orientation of amino acidresidues into the known secondary conformations of proteins. Peptoidsmay also be used which are oligomers of N-substituted amino acids andcan be used as motifs for the generation of chemically diverse librariesof novel molecules.

Peptides of the invention may also be used to identify lead compoundsfor drug development. The structure of the peptides described herein canbe readily determined by a number of methods such as NMR and X-raycrystallography. A comparison of the structures of peptides similar insequence, but differing in the biological activities they elicit intarget molecules can provide information about the structure-activityrelationship of the target. Information obtained from the examination ofstructure-activity relationships can be used to design either modifiedpeptides, or other small molecules or lead compounds that can be testedfor predicted properties as related to the target molecule. The activityof the lead compounds can be evaluated using assays similar to thosedescribed herein.

Information about structure-activity relationships may also be obtainedfrom co-crystallization studies. In these studies, a peptide with adesired activity is crystallized in association with a target molecule,and the X-ray structure of the complex is determined. The structure canthen be compared to the structure of the target molecule in its nativestate, and information from such a comparison may be used to designcompounds expected to possess.

(iii) Drug Screening Methods

In accordance with one embodiment, the invention enables a method forscreening candidate compounds for their ability to increase or decreasethe activity and/or expression of TCAP. The method comprises providingan assay system for assaying TCAP activity, assaying the activity in thepresence or absence of the candidate or test compound and determiningwhether the compound has increased or decreased TCAP activity. Suchcompounds may be useful in treating conditions associated with aberrantregulation of neuronal growth.

Accordingly, the present invention provides a method for identifying acompound that affects TCAP activity or expression comprising:

-   -   (a) incubating a test compound with a TCAP peptide or a nucleic        acid encoding a TCAP peptide; and    -   (b) determining an amount of TCAP peptide activity or expression        and comparing with a control (i.e. in the absence of the test        substance), wherein a change in the TCAP activity or expression        as compared to the control indicates that the test compound has        an effect on TCAP activity or expression.

In accordance with a further embodiment, the invention enables a methodfor screening candidate compounds for their ability to increase ordecrease expression of a TCAP peptide. The method comprises putting acell with a candidate compound, wherein the cell includes a regulatoryregion of a gene encoding TCAP operably joined to a reporter gene codingregion, and detecting a change in expression of the reporter gene.

Such compounds can be selected from protein compounds, chemicals andvarious drugs that are added to the culture medium. After a period ofincubation in the presence of a selected test compound(s), theexpression of mutated TCAP can be examined by quantifying the levels ofTCAP mRNA using standard Northern blotting procedure, as described inthe examples included herein, to determine any changes in expression asa result of the test compound. Cell lines transfected with constructsexpressing TCAP can also be used to test the function of compoundsdeveloped to modify the protein expression.

(f) Therapeutic Uses

As previously discussed, TCAP of the invention is involved in cAMP, cGMPactivity, neuronal growth and neurological development. Accordingly, thepresent invention provides a method of treating a condition associatedwith aberrant regulation of cAMP, cGMP, neuronal growth, neuronalcommunication, or neuronal cell proliferation comprising theadministering to a cell or animal in need thereof, an effective amountof agent that modulates TCAP expression and/or activity.

The term “agent that modulates TCAP expression and/or activity” meansany substance that can alter the expression and/or activity of TCAP.Examples of agents which may be used to in administration include: anucleic acid molecule encoding TCAP; the TCAP peptide as well asfragments, analogs, derivatives or homologs thereof; antibodies;antisense nucleic acids; peptide mimetics; and substances isolated usingthe screening methods described herein that can result in TCAP levelsand/or function consistent with a person without the condition.

The term “effective amount” as used herein means an amount effective, atdosages and for periods of time necessary to achieve the desiredresults.

The term “animal” as used herein includes all members of the animalkingdom that respond to TCAP, preferably mammals, including both humanand non-human animals, more preferably humans. In another embodiment,animals include domesticated animals, such as cows, horses, pigs, andsheep, In another embodiment, the animals are from the avian family andinclude chickens.

In accordance with another embodiment, the present invention enablesgene therapy as a potential therapeutic approach to a condition, inwhich normal copies of the TCAP gene are introduced into patients tosuccessfully code for normal TCAP peptide in several different affectedcell types.

Retroviral vectors can be used for somatic cell gene therapy especiallybecause of their high efficiency of infection and stable integration andexpression. The targeted cells however must be able to divide and theexpression of the levels of normal protein or peptide should be high. ATCAP encoding gene can be cloned into a retroviral vector and drivenfrom its endogenous promoter or from the retroviral long terminal repeator from a promoter specific for the target cell type of interest (suchas lymphoid cells). Other viral vectors that can be used includeadeno-associated virus, vaccinia virus, bovine papilloma virus, or aherpesvirus such as Epstein-Barr virus. Gene transfer could also beachieved using non-viral means requiring infection in vitro. This wouldinclude calcium phosphate, DEAE dextran, electroporation, cationic oranionic lipid formulations (liposomes) and protoplast fusion. Althoughthese methods are available, many of these are lower efficiency.

Anti-sense based strategies can be employed to inhibit TCAP genefunction and as a basis for therapeutic drug design. The principle isbased on the hypothesis that sequence specific suppression of geneexpression can be achieved by intracellular hybridization between mRNAand a complementary anti-sense species. It is possible to synthesizeanti-sense strand nucleotides that bind the sense strand of RNA or DNAwith a high degree of specificity. The formation of a hybrid RNA duplexmay interfere with the processing/transport/translation and/or stabilityof a target mRNA.

Hybridization is required for an antisense effect to occur. Antisenseeffects have been described using a variety of approaches including theuse of antisense oligonucleotides, injection of antisense RNA, DNA andtransfection of antisense RNA expression vectors.

Therapeutic antisense nucleotides can be made as oligonucleotides orexpressed nucleotides. Oligonucleotides are short single strands of DNAwhich are usually 15 to 20 nucleic acid bases long. Expressednucleotides are made using expression vectors such as an adenoviral,retroviral or plasmid vector. The vector is administered to the cells inculture, or to a patient, whose cells then make the antisensenucleotide. Expression vectors can be designed to produce antisense RNA,which can vary in length from a few dozen bases to several thousand.

Antisense effects can be induced by control (sense) sequences. Theextent of phenotypic changes is highly variable. Phenotypic effectsinduced by antisense are based on changes in criteria such as biologicalendpoints, protein levels, protein activation measurement and targetmRNA levels.

(g) Methods and Uses of TCAP for Modulation of Stress Responses, RelatedConditions and Anxiety

The invention also provides a method of detecting an anxiety disorder inan animal by monitoring the effect of TCAP on said animal. If theanxiety response decreases (anxiolytic) as compared to baseline level,than the animal may have a high anxiety related disorder. If the anxietyresponse of an animal increases in response to administration of TCAP,then the animal may have a low anxiety disorder.

The invention provides a method for normalizing the anxiety state of ananimal by administering TCAP to said animal or up-regulating TCAPexpression in said animal.

The invention also provides a method of inducing a desired anxiety statein an animal by:

(a) determining whether the animal is a low or high anxiety animal; and

(b) (i) administering an effective amount of TCAP or TCAP agonist(including a substance or nucleic acid molecule that up regulates TCAPexpression) to increase anxiety in a low anxiety animal and decreaseanxiety in a high anxiety animal; or

(ii) administering an inhibitor of TCAP or TCAP antagonist (including asubstance or nucleic acid molecule, such as a TCAP antisense nucleicacid molecule, that down regulates TCAP expression) to increase anxietyin a high anxiety animal and decrease anxiety in a low anxiety animal.

The invention also provides a method of detecting a modulator of TCAPactivity comprising, administering TCAP to an animal with a knownanxiety state (high or low anxiety), administering the potentialmodulator to said animal and comparing the response to TCAP in thepresence and absence of said substance. If the animal's response to TCAPis different than that of baseline (Animal with TCAP alone, and nosubstance), then said substance is a modulator of TCAP activity. Suchcompounds may be used to treat animals with undesired stress or anxietylevels.

In one embodiment, TCAP is TCAP-1, or analog, derivative or fragmentthereof with similar biological activity.

In another embodiment a modulator of TCAP is administered to modulate orregulate the stress response in an animal.

Stress as used herein is any state that is not homeostasis or metabolicbalance. Stress is also used to refer to the general state of stressorsprovoking stress responses (Sapolsky, 1992). Hoemeostasis refers to thenormal stability of the internal environment (Sapolsky, 1992). AStressor is defined as anything that disrupts physiological balance, beit physical or psychological (Sapolsky, 1992). For example, a stressorin the behavioural experimentals herein (Examples 10 and 11) is definedas a 120 dB tone using the acoustic startle test.

Stress Response as used herein is a physiological or behaviouralresponse to stressor(s). For example, in the behavioural experiments(Examples 10 and 11), stress response is the startle response asmeasured by the acoustic startle testing apparatus (Med Associates, St.Albans, Vt.) following presentation of a 120 dB tone.

Anxiogenic as used herein means a stimulus, internal or external, thatincreases behavioural measures of anxiety in generally accepted tests.In Examples 10 and 11 herein, the behavioural measure of anxiety is thestartle response as measured by the acoustic startle testing apparatus(Med Associates, St. Albans, Vt.) following the presentation of a 120 dBtone. An anxiogenic response is an increase in the startle response.

Anxiolytic as used herein means a stimulus, internal or external, thatdecreases behavioural measures of anxiety in generally accepted tests.In Examples 10 and 11 herein, the behavioural measure of anxiety is thestartle response as measured by the acoustic startle testing apparatus(Med Associates, St. Albans, Vt.) following the presentation of a 120 dBtone. An anxiolytic response is a decrease in the startle response.

Anxiety refers to a generalized state of distress that may be promptedby generalized, non-specific cues, and involves physiological arousal,but often without organized functional behaviour (Lang et al., 2000).Animal models of anxiety attempt to represent some aspect of theetiology, symptomatology, or treatment of these disorders (Menard andTreit, 1999). In the present studies, the acoustic startle response wasused as a measure of anxiety (Frankland of al., 1996, 1997). This testmeasures a simple reflex induced by a loud and unexpected auditorystimulus, and can be measured using standardized equipment (MedAssociates, St. Albans, Vt.).

High Anxiety as used herein means an animal, e.g., rat, that has apost-vehicle injection startle response that is greater than thebaseline response. An average startle response is calculated for thebaseline trials and the post-injection (treatment) test periods. Thetreatment/baseline ratio is then calculated for each animal, e.g., rat.If this ratio is greater than 1, then the animal is classified as highanxiety.

Low Anxiety as used herein means an animal, e.g rat, that has apost-vehicle injection startle response that is less than the baselineresponse. The treatment/baseline ratio is calculated for each animal,e.g. rat, as above. If this ratio is less than 1, then the animal, e.g.rat, is classified as low anxiety.

Normal Anxiety as used herein means an animal, such as a rat that has apost-vehicle injection startle response that is the same as the baselineresponse. The treatment/baseline ratio is calculated for each rat asabove. If this ratio is equal to 1, then the animal, e.g. rat, isclassified as normal anxiety.

(h) The Role of TCAP in the Regulation of Cell Proliferation and in theTreatment of Cancer

In one embodiment, the invention provides a method of regulating cellproliferation by administering an effective amount of TCAP to an animalin need thereof. In another embodiment, the TCAP is administered in vivoor in vitro to decreasing and/or inhibiting cell proliferation. In oneembodiment the cell is cancerous. In another embodiment the cell is aneuronal tumour cell.

In one embodiment, TCAP or modulators thereof can be used in thetreatment of cancer, such as neuroblastomas or other neuronal tumours.

(i) Pharmaceutical Compositions

The above described substances including nucleic acids encoding TCAP,TCAP peptides, antibodies, and antisense oligonucleotides as well asother agents that modulate TCAP activity or expression may be formulatedinto pharmaceutical compositions for administration to subjects in abiologically compatible form suitable for administration in vivo. By“biologically compatible form suitable for administration in vivo” ismeant a form of the substance to be administered in which any toxiceffects are outweighed by the therapeutic effects. The substances may beadministered to living organisms including humans, and animals.

Thus in one embodiment, the invention provides the use of TCAp ormodulator there in the preparation of a medicament for the treatment ofTCAP-related or TCAP regulated medical conditions. For instance, in theregulation of cell proliferation (e.g. cancer), stress, anxiety orneuronal communicative disorders.

Administration of a therapeutically effective amount of pharmaceuticalcompositions of the present invention is defined as an amount effective,at dosages and for periods of time necessary to achieve the desiredtherapeutic result. For example, a therapeutically effective amount of asubstance may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the substance toelicit a desired response in the individual. Dosage regimes may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

An active substance may be administered in a convenient manner such asby injection (subcutaneous, intravenous, etc.), oral administration,inhalation, transdermal application, or rectal administration. Dependingon the route of administration, the active substance may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions that may inactivate the compound. If the activesubstance is a nucleic acid encoding, for example, a TCAP peptide it maybe delivered using techniques known in the art.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985) or Handbook ofPharmaceutical Additives (compiled by Michael and Irene Ash, GowerPublishing Limited, Aldershot, England (1995)). On this basis, thecompositions include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablevehicles or diluents, and may be contained in buffered solutions with asuitable pH and/or be iso-osmotic with physiological fluids. In thisregard, reference can be made to U.S. Pat. No. 5,843,456. As will alsobe appreciated by those skilled, administration of substances describedherein may be by an inactive viral carrier. In one embodiment TCAP canbe administered in a vehicle comprising saline and acetic acid.

(j) Kits

The reagents suitable for carrying out the methods of the invention maybe packaged into convenient kits providing the necessary materials,packaged into suitable containers. Such kits may include all thereagents required to detect a nucleic acid molecule or peptide of theinvention or conjugates of a nucleic acid molecule or peptide of theinvention and another substance, such as a potential modulator of TCAP,and/or the detection of an indicator of TCAP activity, such as cAMP orcGMP, in a sample by means of the methods described herein, andoptionally suitable supports useful in performing the methods of theinvention.

In one embodiment of the invention, the kit includes primers which arecapable of amplifying a nucleic acid molecule of the invention or apredetermined oligonucleotide fragment thereof, all the reagentsrequired to produce the amplified nucleic acid molecule or predeterminedfragment thereof in the polymerase chain reaction, and means forassaying the amplified sequences. In one embodiment, the primers canamplify a nucleic acid encoding a TCAP protein, preferably the proteinof SEQ. ID. NO.: 13, 14, 21, 22, 29, 30, 37, 38, 45, 46, 53, 54, 61, 62,69, 70, 77, 78, 85, 86, 93, 94, 101, 102 or that further has anamidation signal sequence (preferably GKR or GRR), at the carboxyterminus of said peptides, such as 15, 16, 23, 24, 31, 32, 39, 40, 47,48, 55, 56, 63, 64, 71, 72, 79, 80, 87, 88, 95, 96.

The kit may also include restriction enzymes to digest the PCR products.In another embodiment of the invention the kit contains a nucleotideprobe which hybridizes with a nucleic acid molecule of the invention,reagents required for hybridization of the nucleotide probe with thenucleic acid molecule, and directions for its use. In a furtherembodiment of the invention, the kit includes antibodies of theinvention and reagents required for binding of the antibody to a TCAPpeptide of the invention in a sample.

Before testing a sample in accordance with the methods described herein,the sample may be concentrated using techniques known in the art, suchas centrifugation and filtration. For the hybridization and/or PCR-basedmethods described herein, nucleic acids may be extracted from cellextracts of the test sample using techniques known in the art.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example 1 Identification of Teneurin C-Terminal AssociatedPeptide (TCAP)

A. Identification of TCAP mRNA

Cloning of mRNA. A rainbow trout hypothalamic cDNA library wasconstructed as previously described (Barsyte et al., 1999) using aunidirectional vector (Unizap, Stratagene, La Jolla Calif.). A total of600,000 clones were screened using a randomly labelled 305-bp hamsterurocortin cDNA probe (Robinson et al., 1999) [SEQ. ID. NO 120-5′-att caccgccgc tcg gga tct gag cct gca ggc gag cgg cag cga cgg gaa gac ctt ccgctg tcc atc gac ctc aca ttc cac ctg cta cgg acc ctg ctg gag atg gcc cggaca cag agc caa cgc gag cga gca gag cag aac cga atc ata ctc aac gcg gtgggc aag tga tcg gcc cgg tgt ggg acc cca aaa ggc tcg acc ctt tcc cct acctac ccc ggg gct gaa gtc acg cga ccg aag tcg gct tag tcc cgc ggt gca gcgcct ccc aga gtt acc ctg aac aat ccc gc-3′.] Primary, secondary andtertiary screens all utilized the same probe. The size of the clones,positive after the final screen, were determined by restriction analysisthen sequenced using automated Big Dye methods.

Five positive clones were isolated from the rainbow trout hypothalamiclibrary. Of these, one represented a partial sequence of a putativerainbow trout Ten-m3 homologue (FIG. 1). The clone was 2986 bases longcovering the translated portion of 769 bases]. SEQ. ID. NO. 1 shows a756 base portion [SEQ. ID. NO. 2 thereof and a 3′ untranslated region of734 bases. The stop codon and translated portion were identified byalignment with the mouse (accession number AB025412)[SEQ. ID. NO: 132],human (accession number AK027474)[SEQ. ID. NO: 133] and zebrafish(accession number AB026976)) [SEQ. ID. NO: 134], Ten M3 orthologues.Based on the human gene sequence (Locus Link ID#10178) using Locus Linkon the NICB server, the rainbow trout sequence included the terminal 6exons of the gene. The final 3° exon encoded a 251 amino acid residuesequence [SEQ. ID. NO. 3] with a 40-41-residue carboxy-terminal sequence[SEQ. ID. NOS. 13 and 14, respectively] suggestive of a bioactivepeptide. A putative amidation signal was indicated by the GKR amino acidmotif immediately adjacent to the 40-41 residue carboxy terminalsequence and TAA stop codon. 40 residues upstream, a PC-7-like cleavagesignal was present immediately followed by a glutamine suggesting thatthe putative free peptide would begin with a pyroglutamic acid. Thiscleavage site is not necessarily processed in the normal way and cancreate a 40 or 41 amino acid residue mature peptide (starting at 43 or44 amino acid residues upstream from the stop codon).

B. Extraction of Free TCAP Peptide

Tissue Collection: Mouse brains (Mus musculus; n=10; 1.8 g) werecollected and stored at −80° C. for one month, at which time they wereremoved and placed immediately into liquid nitrogen. Brain tissue wascrushed using a mortar and pestle and powdered in the presence of liquidnitrogen.

Activation of C18 packing material: Bondpack® C18 bulk packing material(1 g; 125 Å; 37-55 μm; Waters Corporation, Milford, Mass., USA) wasactivated with 100% methanol (5 ml), vortexed and left to stand (5min.). Excess methanol was removed. C18 was then washed in duplicatewith PBS (5 ml, pH 7.6). An additional PBS aliquot was added (5 ml),vortexed and centrifuged (5000 rpm; 5 min); the supernatant wasdiscarded.

Tissue Extraction: Acetonitrile (90%) and TFA (0.05%) were added topowdered brains in a 5:1 volume to weight ratio, mixed for 1 hr on analiquot mixer rocker. The mixture was centrifuged (8000 rpm×20 min.);the supernatant was removed and saved. The remaining solids wereback-extracted in acetonitrile (90%) and TFA (0.05%) in 40% of thesolvent volume used in the initial extraction, vortexed and centrifugedas described previously. The supernatants were pooled and combined withactivated C18 packing material, vortexed, mixed (1 hr) and centrifuged(8000 rpm×10 min). The supernatant was discarded while the pellet wassubjected to three successive, independent acetonitrile extractions of20%, 50% and 90% respectively. Acetonitrile (5 ml) was added to thepellet, vortexed, mixed (20 min) and centrifuged (6000 rpm×10 min.).Resulting supernatant was saved and concentrated to 800 μl on a vacuumconcentrator (Brinkman Instruments) for HPLC analysis while the pelletwas re-extracted in the same manner.

HPLC Purification of Free TCAP in Brain Extracts

A Beckman model 126 HPLC System Gold (Beckman, Palo Alto, Calif.),attached to a UV detector module 168 and C18 column (3.5 um particlesize; Waters Inc) was used to purify the TCAP peptide extracted frommouse brains (n=10).

A single injection (800 ul) was applied to the column through a 1 mlinjection loop and carried to the column at a flow rate of 1 ml/minusing a dual solvent system (A: 0.05% trifluoroacetic acid (TFA); B: 80%acetonitrile, 0.05% TFA). Following an initial isocratic period of 10min, mobile phase B was increased from 10% to 60% over 75 min, heldisocratically for 5 min and returned to 10% over 5 min. Fractions werecollected (1 ml/fraction), aliquoted (500 ul) and concentrated to 50 ulfor analysis using mass spectrometry.

Example 2 Detection of the Cleaved TCAP in Cell and Tissue Extracts

HPLC as described in Example 1 can be used to detect TCAP. MassSpectroscopy can also be used. Other detection methods can also becombined with HPLC, Mass Spectroscopy or used on their own, such asradio immunoassays, ELISAs, capillary electrophoresis,immunofluorescence confocal microscopy. Mass spectrometric methodsidentify molecules on the basis of a charged molecule's (ion) mass tocharge ratio. A precise determination of the molecules mass is thendetermined allowing for identification of the molecule. Larger peptidescan be sequenced by subsequent fragmentation of the peptide in acollision chamber. This causes preferential breaking of the peptidebonds. The amino acid and peptide fragments are identified by their massto charge ratio. Radioimmunoassays or enzyme-linked immunosorbant assays(ELISA) utilize an antiserum specific for the molecule of interest. Themolecule (TCAP) competes with a tagged structurally similar referencemolecules to bind the antibody. The bound and unbound fractions areseparated from each other and the quantity of remaining tagged TCAP ismeasured. This measurement is proportional to the amount of unlabeledTCAP present. Capillary electrophoresis can also be used to identifyTCAP using an antibody reaction. In this method, the unbound componentis separated from the bound component by migration in an electric field.Immunofluoresence confocal microscopy utilizes a specific antibody boundto TCAP and a secondary antibody that binds to the primary antibody. Thesecondary antibody is effectively conjugated to an enzyme that catalyzesa fluorescent reaction upon introduction of the appropriate substrate.The amount of fluorescence is proportional to the amount of TCAP and ismeasured using digital image analysis.

Mass Spectrometry Detection of Peptide

Samples were dissolved in 5 ul of 1:1 (vol/vol) Acetonitrile:water (plus0.1% (vol/vol) formic acid). Typically, 2-3 ul of each sample was loadedon a glass capillary probe tip and analyzed on a Micromass Q-TOF (hybridquadrupole time of flight) mass spectrometer (Micromass, Manchester,UK). All spectra were acquired under nanospray, positive-ion mode. ForMS measurements the quadrupole RF value was set at 0.5. The scanningregion (m/z) was between 200-2000 with a scan time of 1 s and a dwelltime of 0.1 s. The data was analyzed using MassLynx program (Micromass,Manchester, UK).

Example 3 Synthesis and Solubilization of Peptide

Rainbow trout TCAP-3 [SEQ. ID. NO: 13], wherein the terminal isoleucine(I) was amidated [to give SEQ. ID. NO. 15] was synthesized on anautomated peptide synthesizer, Model Novayn Crystal (NovaBiochem, UKLtd. Nottingham, UK) on PEG-PS resin using continuous flow Fmocchemistry (Calbiochem-Novabiochem Group, San Diego, Calif.). Eight timesexcess diisopropyl ethy amine (Sigma Aldrich Canada Ltd) and four timesexcess Fmoc-amino acid activated with HATU(O-(7-azabenzotriazol-1-,3,3-tetramethyluronium hexfluorophosphate,Applied Biosystems, Foster City, Calif.) at a 1:1 (mole/mole) ratio wereused during the coupling reaction. The reaction time was 1 hour. Asolution of 20% piperidine (Sigma-Aldrich Canada Ltd) inN,N-dimethylformide (DMF; Calcdon Laboratories Ltd, Canada was used forthe deprotection step in the synthesis cycle. The DMF was purifiedin-house and used fresh each time as a solvent for the synthesis. Thecleavage/deprotection of the final peptide was carried out withtrifluoroacetic acid (TFA), thioanisole, 1,2 ethandithiol, m-cresole,triisopropylsilane, and bromotrimethyl silane (Sigma-Aldrich Canada Ltd)at a ratio of 40:10:5:1:1:5. Finally, it was desalted on a Sephadex G-10column using aqueous 0.1% TFA solution and lyophilized. The peptidestructure was confirmed by reverse-phase HPLC, amino acid analysis andatmospheric pressure ionization mass spectrometry. The HPLC and Massspectrometry can be done as described in Examples 1 and 2 herein. Seeabove method. The same method was used to synthesize mouse TCAP-1.

The peptide was solubilized using a number of different methods,however, the best results were obtained using alpha cyclodextrin. Aceticacid (1 ul) was added to dry TCAP at room temperature, vortexed and leftto stand (30 min). Alpha-cyclodextrin (company) was then added in a 4:1volume to dry weight ratio (0.25 ug/ul), vortexed, and concentrated to10% of the original volume on an Eppendorf Vacufuge at 30° C. for 2 hand room temperature for the remainder of the process. Distilled,de-ionized water and physiological saline were then added independentlyin a 1:1 and 3:1, volume to concentrated volume ratio respectively. Thissolution (0.5 ug/ul) was vortexed and centrifuged (11,000 rpm; 3 min).The supernatant was aliquoted and stored at 4° C. The same method wasused to synthesize and solubilize other TCAPS including mouse TCAP-1.

Example 4 Peptide Sequence Relationships and Phylogeny

The rainbow trout Teneurin 3 exon including the TCAP portion shows ahigh degree of conservation among its orthologues in zebrafish, mouse,and humans (FIG. 2). However the trout sequences also showed highsequence similarity with four mouse Teneurin protein paraloguesdesignated as Teneurin 1 to 4 (FIG. 3) and similarly four humanparalogues were found in the sequence data base (FIG. 4). All possess ahigh degree of similarity among members of the protein family. TheTeneurin protein family represents a type II transmembrane protein wherethe carboxy terminus is displayed on the extracellular face of theplasma membrane (FIGS. 6 A and B). The TCAP portion represents only theC-terminal residues of the protein. The TCAP sequence is highlyconserved across vertebrate species and even the Drosophila versionpossesses about 60% sequence identity (accession number AF008228) (FIGS.7A and B).

FIG. 5 illustrates the preTCAP nucleotide coding sequences for human,mouse, zebrafish and rainbow trout plus the stop codon. The codingsequences for TCAP (40 and 41 amino acid residue sequences) can beeasily determined from the figure.

A comparison of the conserved motifs within the primary structure of theTCAP and CRF families show a match (FIG. 9). Conserved motifs ofI/L-S-X-X (X)-L/V [SEQ. ID. NO: 129] at the amino terminus,LN-L/I-X-V/aliphatic residue [SEQ. ID. NO: 130] in the middle and themotif N-I/A-H/basic residue-I/L/F-aliphatic residue [SEQ. ID. NO: 131]at the carboxy terminus. A more compelling gage of similarity, however,is shown by the secondary structure predictions (FIG. 10). TCAP shows ahighly similar polarity profile in comparison to others in the peptidesuperfamily. Hydrophobicity, using a Kyte-Doolittle plot shows a generalsimilarity within the middle and carboxy terminal regions, but a morehydrophobic amino terminal region.

Although CRF and urocortin show high sequence similarity for each otherand urocortin 2 and 3 show high similarity, the level of identitybetween these two paralogous lineages is only about 11%. The level ofidentity among TCAP members is about 60% (FIG. 8). CRF and TCAP belongto a much larger peptide family that also includes the insect diureticpeptides (FIG. 11). Key motifs, outlined in FIG. 9 show alignment whenthe insect diuretic peptides are included.

Example 5 PCR Expression of Teneurin mRNA

The presence of the Teneurin protein in brain extracts and on cell lineswere established using PCR. Primers utilized in this experiment weredesigned from 3′-ends of the published sequences for mouse Ten-M 1, 2,3, and 4 [SEQ. ID. NOS: 4-7]. The TCAP-1 forward primer (25mer:5′-ACGTCAGTGTTGATGGGAGGACTA-3′)[SEQ. ID. NO: 121] is complementary tonucleotides 7938-7962 of Teneurin 1. The Teneurin 1 reverse primer(27mer: 5′-CCTCCTGCCTATTTCACTCTGTCTCAT-3′) [SEQ. ID. NO: 122] isspecific for nucleotides 8262-8288 of Teneurin 1. The primers werepredicted to generate a Ten-M1 PCR product of 351 bps. The Teneurin 2forward primer (25mer: 5′-TCGAGGGCAAGGACACACACTACTT-3′) [SEQ. ID. NO:123] is complementary to nucleotides 7920-7944 of Teneurin 2. TheTeneurin 2 reverse primer (26mer: AAGAACTGGATGTTGCTGCTACTGTC-3′) [SEQ.ID. NO: 124] is complementary to nucleotides 8354-8379 of Teneurin 2.The primers were predicted to get a Teneurin 2 PCR product of 460 bps.The Teneurin 3 forward primer (25mer: 5′-CAACAACGCCTTCTACCTGGAGAAC)[SEQ. ID. NO: 12]5 is complementary to nucleotides 7681-7705 of Teneurin3. The Teneurin 3 reverse primer (21mer: 5′-TGTTGTTGGCACTGTCAGCCA-3′)[SEQ. ID. NO: 126] is specific for nucleotides 8139-8159. The predictedPCR product for Teneurin 3 primers is 479 bps. The Teneurin 4 forwardprimer (23mer: 5′-TTTGCCTCCAGTGGTTCCATCTT-3′) [SEQ. ID. NO: 127] iscomplementary to nucleotides 7868-7890 of Teneurin 4. The Teneurin 4reverse primer (24mer: 5′-TGGATATTGTTGGCGCTGTCTGAC-3′) [SEQ. ID. NO:128] is complementary to nucleotides 8446-8469 of Teneurin 4. Theprimers were predicted to generate a Teneurin 4 PCR product of 602 bps.

The total RNA of Gn11 cells was isolated using RNeasy Mini Kit (Qiagen).First strand synthesis was performed by using First-Strand Beads(Amersham Pharmacia Biotech). Briefly, 2 μg of total RNA was mixed withthe first strand reaction beads (include buffer, dNTPs, murine reversetranscriptase, RNAguard, and RNase/DNase-free BSA) and 0.2 μg randomhexamer pd(N)₆ in a volume of 33 μl. Extension was carried out for 60minutes at 37° C.

The PCR for Teneurin 1, 2, 3, and 4 was performed respectively using 1μl cDNA with a final reaction volume of 50 μl containing 0.2 mM eachdNTP, 5 μl 10× buffer, 1.5 mM MgCl, 1 ul Taq DNA polymerase, 0.2 μM eachTeneurin primer and 0.1 μM each GAPDH primer (forward and reverseprimers; The expected GAPDH DNA≈200 bps). The initial denaturation wasset over an interval of 3 min at 94° C. After 35 cycles of 1 min. at 94°C., 1 min. at 60° C., and 1 min. at 72° C., a 5 min. extension wasperformed at 72° C. The PCR products were examined by 1.5% agarose gelelectrophoresis. The appropriate size DNAs of Teneurin 1, 2 and 4 wereextracted from the gel using DNA extraction kit (MBI-Fermentas). TheTeneurin 1, 2 and 4 DNAs recovered from the gel were subcloned by usingthe TOPO TA Cloning kit (Invitrogen Corporation). Briefly, the pCR®2.1-TOPO plasmids with Teneurin 1, 2 or 4 DNA were transformed intochemically competent E. coli and cultured on LB agar plates and inliquid LB medium successively. The products were purified by using thePerfectprep Plasmid Midi Kit (Eppendorf). Positive results were selectedby digesting the plasmids using the restriction endonuclease EcoRI andthen by electrophoresis. The positive plasmids were sequencedcommercially using T7 sequencing primer (AGTC Corp, Toronto, Canada).

Results

A positive amplification product was obtained from adult mouse cells forTeneurin 1, 2 and 4 using PCR (FIG. 12). Similarly, the same productswere obtained using mRNA extracted from the immortalized neuronal line,Gn11. A neuronal cell line isolated from the same tumour, NLT, showedexpression of only Teneurin 2 and 4. However, a neuroblastoma cell line,Neuro2a appeared to express all four forms of the Teneurin gene family.The Neuro2a is the least differentiated of the cell lines used. A ratfibroblast cell line, TGR1, also showed the presence of paralogues 1, 2and 4 (data not shown). The identity of the amplication signal wasconfirmed by sequence analysis. TCAP-1 primers generated a 351 bpssequence and showed 99.43% coincidence with Teneurin 1 DNA. TCAP-2primers generated a 455 bps sequence and showed 99.56% coincidence withTeneurin 2 DNA. TCAP-4 primers generated a 602 bps sequence and showed99.83% coincidence with Tenuerin 4 DNA. The TCAP 3 primers amplified a306 bp sequence from mouse neuroblastoma Neuro2a cells. The amplifiedsequence possesses a 173-bp deletion upstream of the TCAP cleavagesignal. This finding indicates that the TCAP-3 primers are specific, butthat the Neuro2a cells appear to possess a variant of Teneurin 3.

Example 6 Cell Proliferation Experiments

Several cell lines were utilized initially to establish a model systemfor which the TCAP could be evaluated. Initially the mouse neuroblastomacell line, Neuro2a, the human breast cancer cell line MCF-7, mouseGnRH-secreting immortalized neuron lines NLT and Gn11 COS-7 cells, andthe rat fibroblast cell line TGR1. Preliminary studies indicated thatthe cells were responsive to the effects of TCAP Rainbow Trout TCAP-3,SEQ. ID. NO: 13: amidated [SEQ. ID. NO. 15], in that the cells showed adecrease in cell proliferation (data not shown). The studies wereperformed essentially in accordance with the cell proliferation studiesbelow. Gn11 and TGR1 cells were selected to be used for further studies.

Pharmacological Test of TCAP on fibroblast Cell Lines TGR1 and HO16.4c:2 plates containing 3×10⁴ TGR1 cells/well and 2 plates containing 3×10⁴HO16.4c cells/well in full-serum medium were prepared for testing. Each6-wells in the plate was designed as a testing group. 24 hours later,aliquots (20 μl) of drugs) were added in a 12-hours interval afterchanging the medium using fresh full-serum DMEM. The cells were observedthrough a microscope per 4-hours. The numbers of the two cell lines werefound significantly lower in TCAP groups at 48-hrs and 72-hrs stages.Cells were counted at 48 hours and 72 hours after being treated. Twoplates containing 3×10⁴ Gn11 cells/well in full-serum medium wereprepared for testing. Each 6-wells in the plate was designed as atesting group. 24 hours later, aliquots (20 μl) of drugs (vehicle:saline+acetic acid; 10⁻⁶ M TCAP-3) were added in a 12-hours intervalafter changing the medium using fresh full-serum DMEM. The cells wereobserved through a microscope per 4-hours. Cells were counted at 48hours and 72 hours after being treated.

A concentration of 10⁻⁶ M of TCAP administered at 0, 12 24 and 36 hoursdecreased the proliferation of a mouse neuronal cell line (Gn11) (FIG.13A—48 hrs and 13B—72 hrs), a rat fibroblast cell line (TGR1) by 50-60%at 48 hours (FIG. 14) and a HO16.4c cells at 48 hours relative to thevehicle treated cells (FIG. 15).

The ability of TCAP to inhibit cell proliferation in the above-notedcell lines, indicates that the peptide would be useful in theregulations of cell proliferation and associated medical conditions suchas in the treatment of cancer TCAP could be used to arrest tumour growthand inhibit metastasis. In a preferred embodiment, TCAP could be used inthe treatment of neuronal tumors.

Example 7 Cyclic Nucleotide Experiments

I. A. cAMP and cGMP Assays

Approximately 10⁶ Gn11 cells were treated with 20 uL of 10⁻⁹, 10⁻⁸, or10⁻⁷ or 10⁻⁶M TCAP-1 or TCAP-3 and incubated at 37 C for 10 minutes.Medium and peptide was removed and the cells were lysed using 350 uL ofa 0.1 M HCL 0.1% Triton X-100 solution. Using the same concentrated HCland Triton X-100 solution and a provided standard concentrate, fivestandard solutions were made up with concentrations of 200, 50, 12.5,3.12 and 0.78 μmol/ml. All reactions were done in triplicates. Wellswere set up for blanks, non-specific binding, total activity (TA), zerobinding, five standards, and 12 samples. Using a 96-well IgG coatedplate, 50 uL of neutralizing reagent were pipetted into each well exceptthe blanks. 150 uL of the 0.1 M HCL/0.1% Triton solution was pipettedinto the NSB wells and 100 uL of this solution was pipetted into thezero binding wells. 100 uL of the standards and 100 uL of the sampleswere pipetted into their respective wells. 50 uL of conjugate werepipetted into each well except the TA and the blank wells. 50 uL of thecAMP antibody were pipetted into each well except the TA, blank and NSBwells. The plate was allowed to shake overnight. The following morning,the wells were rinsed three times with a 10 times diluted wash buffersolution. 50 uL of conjugate was added to the TA wells and 200 uL ofp-Npp substrate was added to each well. The plate was covered again andincubated at room temp for one hour. At this point, 50 uL of stopsolution was added to all wells and the absorbance was read at 405 nmusing a Spectramax spectrophotometer. Three levels of controls wereutilized: A blank tube which provides a measure of any reactivitybetween p-Npp substrate and IgG coated wells; TA: measure of activity ofalkaline phosphotase in conjugate, if any; NSB: measure of binding ofconjugate to plate or to antibody; Bo: measure of binding conjugate toantibody (no sample and conjugate competition).

B. Results

In the first set of experiments, Gn11 cells were treated with 10⁻⁶ M ofrtTCAP-3 SEQ. ID. NO:13, amidated [SEQ. ID. NO: 15], see above, raturocortin or the vehicle, as above (FIG. 16A). TCAP reduced cAMPaccumulation in these cells to 58.9±4.8% of the vehicle-treated cells(p<0.01). Urocortin induced a non-significant decrease of 89.2±6.3% ofthe control cells. In cGMP accumulation experiments, TCAP reduced cGMPaccumulation to 38.5±8.8% of the control cells (p<0.01) whereasurocortin caused a decrease to 50.0±8.5% of the control cells. (FIG.16B)

II. A. cAMP Assays

Gn11 cells were treated when the confluence reached 70%. The cells weretreated with 10⁻⁹, 10⁻⁸ or 10⁻⁷M TCAP, urocortin and vehicle,separately, and incubated in incubator at 37° C. (Details below) Mediumwas removed and the cells were washed by PBS one time, and then werelysed using 600 uL of 0.1 M HCL solution. After freezing/thawing 3times, the samples were transferred into microcentrifuge tubes. At thesame time, squeezed the cells by 3 ml syringe and 22 G needle 20 times.Centrifuge 4000 rpm×5 min, the supernatant of each sample was aspiratedand kept in the −20° C. freezer until the cAMP or cGMP assay was carriedon. Using the same concentrated HCl and a provided standard concentrate,five standard solutions were made up with concentrations of 200, 50,12.5, 3.12 and 0.78 μmol/ml. All reactions were done in duplicates.Wells were set up for blanks, non-specific binding (NSB), total activity(TA), zero binding (BO), five standards, and all samples. Using a96-well IgG coated plate, 50 uL of neutralizing reagent were pipettedinto each well except the blanks and TA. 150 uL of the 0.1 M HCL waspipetted into the NSB wells and 100 uL of this solution was pipettedinto the zero binding wells. 100 uL of the standards and 100 uL of thesamples were pipetted into their respective wells. 50 uL of conjugatewere pipetted into each well except the TA and the blank wells. 50 uL ofthe cAMP antibody were pipetted into each well except the TA, blank andNSB wells. The plate was allowed to shake overnight (18 h) at 200 rpm at4° C. The next day, the wells were rinsed three times with a 10 timesdiluted wash buffer solution. After each well was dried thoroughly, 5 uLof conjugate was added to the TA wells and 200 uL of p-Npp substrate wasadded to each well. The plate was covered again and incubated at roomtemp for one hour without shaking. At this point, 50 uL of stop solutionwas added to all wells and the absorbance was read at 405 nm and 580 nmusing a Spectramax spectrophotometer. The data of 580 nm were providedthe background of each well, which were subtracted from the data of 405nm.

B. Results

10⁻⁸ M TCAP induced a significant increase in cAMP accumulation at 15minutes after introduction of the peptide and fell to normal limitswithin 30 minutes of treatment (FIG. 17A). Urocortin was used for thepurpose of a positive control. FIG. 17B illustrates cAMP levels in Gn11cells in the presence of 10⁻⁴ M 3-isobutyl-1 methyl xanthine (IBMX), aphosphodiesterase inhibitor used to boost cAMP induced by treatment of10⁻⁸ MTCAP or urocortin. FIG. 17C is a bar graph illustrating cAMPaccumulation over 30 minutes in Gn11 cells by administration of variousconcentrations of TCAP or Urocortin in the presence of IBMX. FIG. 17D isa bar graph illustrating inhibition of 10⁻⁸ M forskolin-stimulated cAMPby 10⁻⁸ MTCAP or urocortin.

Example 8 Behavioural Studies A. Brain Stimulation Reward BehaviourExperiments

Rats can be trained to bar press for electrical stimulation of thelateral hypothalamus which activates cholinergic nuclei of the pontinetegmentum and their projections to dopaminergic paths of the forebrain.Once reliable baseline rates of bar pressing have been established for agiven current, the consequences of various drugs for the activity ofthis cholinergic dopaminergic system can be assessed by makinginjections of substances intracranially and then observing their effectson rates of self stimulating behaviour. TCAP-3 SEQ. ID. NO: 13,amidated, [SEQ. ID. NO. 15] see above, at a concentrations of 1 nMprepared in physiological saline was injected by canulae into thelaterodorsal tegmental nucleus through guide cannulae. The rate of barpressing was compared to the vehicle treated rats.

B. Results

A robust inhibition of self-reward stimulus occurred when TCAP at 1 nM(4.2 pg/ul) was injected into the caudal midbrain of rats (FIG. 18). Inboth forebrain (lateral ventricle) and midbrain injections the effectwas reversible with the rats behaviour returning to normal limits afterabout 60 minutes.

Example 9 Preliminary In Situ Hybridization Results

The first in situ hybridization data indicate that the Teneurin I gene(TCAP-1) is highly expressed in adult rat brain. The regions of greatestexpression occur in the lateral septum, bed nucleus of the striaterminalis ventral medial nucleus of the hypothalamus and ventralpremammalary nucleus. Lesser expression occurs in the hippocampus andamygdala. This expression pattern is consistent with peptides regulatingthe stress response (see above) in emotional and mood disorders. Thesedata indicate that TCAP plays a primary role in stress and anxietyregulation rather than one of neurogenesis and neurodegeneration. TheTeneurin 4 (TCAP-4) expression also occurs in the adult brain butTeneurin 1 is stronger.

A. Methods

The methods were performed as previously described (Simmons et al.,1989; Ericsson et al., 1995) using ³⁵S-labelled antisense and sense(control) probes higher high stringency conditions (50% formamide withfinal washes at 0.2 SSC at 60 C). The ³⁵S-labelled cRNA probes weregenerated from 350 bp cDNA of exon 33 including the TCAP portion by invitro transcription with the appropriate polymerases (T3 for antisenseand T7 for sense).

B. Results

Results are shown in FIG. 20. On the left column is the expression ofTCAP-1 mRNA using the antisense probe, and on the right column, thesense probe. A-B. central nucleus of the amygdala (CeA); C-D. bednucleus of the stria terminalis, medial (BSTM); E-F: premammilaryventral nucleus (PMV). Abbreviations: 3V, third ventricle; fx, formix;ic, internal capsule; LV, lateral ventricle; MeA, medial nucleus of theamygdala; opt, optic tract; st, stria terminalis. Bars=300 μm (A-B) and500 μm (C-F).

The in situ hybridization data indicate that the TCAP-1 gene is highlyexpressed in adult rat brain. The expression of the C-terminalteneurin-1 exon including the TCAP-1 region was restricted tohypothalamic and limbic regions (FIG. 20 A-F). The regions of greatestexpression occur in the lateral septum, bed nucleus of the striaterminalis ventral medial nucleus of the hypothalamus and ventralpremammalary nucleus. Lesser expression occurs in the hippocampus andamygdala. This distribution is consistent with TCAP playing a modulatoryrole with emotionality, anxiety and motivation. The presence of TCAP-1expression in the ventral premammillary nucleus is of particularinterest as there are no known CRF receptors found in this region (Li etal., 2002). There was no evidence that the TCAP containing exon wasexpressed in regions associated with neurogenesis, such as the olfactorylobes or subependymal layers of the lateral ventricles. Despite theprevious recognition of the teneurin proteins, their expression in adultbrain has never been examined. However, teneurin 1 and 4 expression hasbeen observed in the diencephalon of developing mouse, chick andzebrafish brain (Rubin et al., 1999; Ben-Zur et al, 2000; Mieda et al.,1999).

These data support the hypothesis that TCAP primary role is one ofstress and anxiety regulation.

Example 10 Chronic TCAP Study: The Role of TCAP in Modulating the StressResponse A. Method

-   -   1. Wistar Rats were tested in acoustic startle for baseline        response (1 hour test consisting of 60 acoustic startle stimuli,        120 dB, 60 sec inter-stimulus interval), and divided into        matched groups to receive either TCAP-1 (10 nmol of mouse        TCAP-1, amidated [SEQ. ID. NO. 40] in 30 vehicle        intra-cerebroventricularly) or Vehicle (e.g. saline and acetic        acid).    -   2. Two days later, rats were tested in acoustic startle, 25        stimuli baseline (120 dB, 60 sec inter-stimulus interval), then        injected ICV with 10 nmol TCAP-1 or Vehicle, then acute response        was measured for 1 h (60 stimuli, 120 dB, 60 sec inter-stimulus        interval).    -   3. 25 days later, rats were given either TCAP-1 (10 nmol in 3 μl        or vehicle (3 μl once per day for 5 consecutive days ICV.    -   4. Rats were left alone for 10 days.    -   5. On the 10^(th) day, rats were tested for acoustic startle        response without TCAP-1.

On the 11th day, rats were re-tested for startle response, again withoutTCAP-1, for 60 minutes (60 stimuli, 60 sec inter-stimulus interval, 120dB). Re-tested in startle 13^(th) and 28^(th) days. The vehicle is themixture of saline and acetic acid into which TCAP-1 was dissolved. Whenreferring to vehicle, this refers to the solution without the additionof TCAP-1.

B. Results

Results are shown in FIG. 21 for the 0, 10 and 12 days after the 5consecutive day ICV of Vehicle (21A) or TCAP-1 (21B). Startle responsesfor animals in the chronic study are shown in FIG. 22. The averagestartle response for the two groups (TCAP-1 and Vehicle) on Day 1,before chronic TCAP treatment is shown in FIG. 22A. FIG. 22B shows theaverage startle response for TCAP and vehicle groups over the 60 trialsin the session on the 10^(th) day after chronic TCAP treatment. FIG. 22Cshows the mean baseline startle responses for all animals for TCAP andvehicle groups averaged across all 60 trials.

Example 11 Acute TCAP Study Acoustic Startle Measurements A. Method

Male Wistar rats (250-275 g), were surgically implanted with cannulae(23 gauge) bilaterally into the basolateral nuclei of the amydala (AP−2.8, ML +/−5.0, DV −7.2 mm, from bregma). One week later, the animalswere habituated to the acoustic startle reflex (ASR) chambers (MEDAssociates, grid rod cage measuring 7.5″×3.6″×4.2″), consisting of 25trials of 120 dB stimuli presented randomly with an inter-stimulusinterval of 55-65 seconds, duration of 30 msecs and frequency of 5000Hz. The same stimulus conditions were used for test days, whichconsisted of a 25 trial baseline, injection with mouse TCAP-1 (withamidation signal)[SEQ. ID. NO. 40] or vehicle (0.25 μl/side, flow rate0.5 μl/min), and testing for a further 60 trials post-drug. Each ratreceived vehicle treatment on the first test day then TCAP-1 (e.g.mouse. TCAP-1) in a random and counter balanced fashion in subsequenttest days, spaced 48 h apart. On the final test day, all rats againreceived vehicle treatment. Following histological analysis of cannulaeplacements, the data of eight rats was retained for statisticalanalysis.

From the data, rats were divided into high and low anxiety groupsdepending upon their treatment/baseline ratio for the vehicle. Animalsthat scored less than one were considered low anxiety, those scoringmore than one were considered high anxiety. There were four animals ineach anxiety group.

Results are shown in FIGS. 23 and 24. FIG. 23 is a bar graphillustrating the mean treatment/baseline value for both groups for allconcentrations of mouse TCAP-1. A repeated measures ANOVA indicated thatthe level of significant differences between the two anxiety groups wasP=0.0078. After TCAP-1 treatment the treatment/baseline ratio of lowanxiety was similar to the initial high anxiety value and vice versa. Avehicle injection was performed at the end of the study to show that theeffect was due to the TCAP-1 and not to the experience of injection.TCAP 1 concentrations were 3, 30, 300 pmoles. A summary of the effect ofamygdala-injected TCAP-1 is illustrated in FIG. 24. It was shown thatthe effect by TCAP-1 on startle response is inversely proportional tothe baseline startle response. As such TCAP-1 can be used to normalizestartle behaviour or stress response.

Discussion

Regardless of the mechanism the synthetic TCAP peptide is potent, invivo at eliciting a behavioural response in rats. Given the strongexpression of TCAP in hypothalamic and limbic regions, the syntheticmouse TCAP-1 peptide with amidation signal was micro injected into thebasolateral amygdala to determine effects on acoustic startle in rats.Animals possessing a high treatment-to-baseline ratio (>1) showed asignificant (p<0.05) decrease in startle magnitude, whereas animals witha low treatment-to-baseline ratio (<1) showed a significant (p, 0.05)and does dependent increase in startle magnitude (FIG. 23). These dataindicate that TCAP-1 acts to modulate the effect on startle responsesdepending on baseline reactivity of the particular animal and cannormalize the behaviour associated with acoustic startle. Otherneuropeptides that have been demonstrated to increase acoustic startleare CRF (Liang et al., 1992), CCK (Frankland et al., 1997) and SP (Kraseet al., 1994/1999). The acoustic startle paradigm is a well-known andextensively used paradigm for assessing the anxiogenic or anxiolyticeffects of drugs. This is an ideal paradigm for testing a novel compoundsince the startle reflex does not involve locomotion, learning, memory,or motivated behaviour of any kind, which could possibly confound theinterpretation of the results.

The data presented indicate that TCAP represent a new family ofneuropeptides associated with the regulation of anxiety by regulatingneuronal function in key regions of the forebrain and limbic system.Previous studies have also suggested a role of the teneurin genes withneural regulation. Human Ten-M1 maps to position Xq25 of the Xchromosome (Ben-Zur et al., 1999). This is a region associated withX-linked mental retardation syndromes (Minet et al., 1999). Theconditions mapped to this site are characterized by severe mentalretardation and may include motor sensory neuropathy, deafness andsometimes seizures and impaired vision.

The regulation of TCAP represent a new target to understand theaetiology of neurological dysfunction and psychiatric illness. Theexample shows that TCAP can be used in the treatment of stress-relateddisorders and in other neuropathological conditions.

Example 12 Activity of TCAP on Immortalized Neurons A. In Vitro Assays

Gn11 immortalized neurons were cultured as previously reported (Tellamet al., 1998) Direct cAMP measurements were performed with thenonacetylated version of a commercial kit (Assay Designs, Ann Arbor,Mich.). After starved by serum-free DMEM for 1 hr and replaced withfresh DMEM without serum, cells were treated for 15 min with TCAP,urocortin or vehicle ±CRFR1 antagonist PD171729 in the continuedpresence of forskolin (1 μM) and IBMX (100 μM. Protein assays: Totalprotein was determined using the BCA protein assay method (Pierce Co).MTT Assays: Gn11 cells were seeded into 96-well plates and cultured infull serum DMEM until the cells were 30% confluent. Vehicle, 1 nM, 10 nMor 100 nM TCAP-1 were added into each group (n=8). (FIG. 25A) The MTTassay (Sigma Chemicals) was performed at 0, 6, 12, 24 and 48 hours. FlowCytometry: DNA content of the Gn11 cells was quantified by staining withpropridium iodide and analyzed on a FACSCAN flow cytometer (BeckmanInstruments).

B. Results

Mouse TCAP-1 induced a dose-dependent change in cAMP accumulation inmouse immortalized neurons after 15 minutes. A 1 nM dose increased(p<0.05) cAMP levels 45% over the vehicle-treated cells. In contrast,100 nM TCAP-1 decreased (p<0.05) cAMP accumulation 40% from controlcells (FIG. 25A). However, co-treatment with the specific CRF type 1receptor antagonist, PD171729 failed to completely abolish TCAP'seffects at cAMP accumulation. In contrast, the same concentration ofantagonist induced a complete inhibition (p<0.01) ofurocortin-stimulated cAMP accumulation in these cells (FIG. 25B). Wehave previously established that these cells possess a CRF-R1 receptor(Tellam et al., 1998) but not an R2 receptor (data not shown).Concentrations of 1, 10 and 100 nM of TCAP-1 induced a significantincrease in total protein concentration after 120 minutes in the samecells (FIG. 25C). Mouse TCAP-1 treatment of these cells also induced adose-dependent effect on cell metabolism. Cellular activity as indicatedby mitochondrial activity (MTT assay) showed a significant (p<0.05)increase in activity at 1 nM concentration, but a decrease at 100 nMconcentrations (FIG. 25D). Similarly, 1 nM TCAP reduced (p<0.05) theincidence of G1 phase after 24 hours whereas a 100 nM dose increased(p<0.05) G1 phase as determined by DNA content analysis.

As such α-helical CRF(9-41) antagonist can modulate TCAP stress responsemodulating activity.

Example 13 Proteomic Profiling and MicroArray Studies

To determine the effect of TCAP and to develop a cell model system forscreening TCAP modulators, diagnostic and conditions related to TCAP andmethods of medical treatment, TCAP responsive cell lines were subject toproteomic profiling and microarray analysis. This was done using anon-tumorgenic-derived immortalized murine hypothalamic cell line, N38,which has the marker profile shown in Table 1. The effect of TCAP onother immortalized cell lines can be preformed by adapting the methodnoted below.

A. TCAP Responsive Immortalized Hypothalamic Cell Lines

The TCAP responsive immortalized cell lines used were prepared by DeniseBelsham, University of Toronto, by preparing a culture of embryonichypothalamic cells; infecting said culture with a retrovirus encoding aviral oncogene, large T Antigen, operably linked to a promoter and aselectable marker; isolating transfected cells from non-transfectedcells to obtain a culture of immortalized hypothalamic cells; subcloningsaid immortalized cells into sub-cloned populations; and screening saidsubcloned populations for expression of specific neuronal markers; andselecting and further cloning a specific population. The immortalizedcell lines can then be screened for TCAP responsiveness.

TCAP responsiveness was screened by measuring the functional cAMPresponse of the immortalized subclones to TCAP. The results are shown inFIG. 26. N-15-1, #7 (N7), N-18-1, #11 (N22), and N-15-14, #29 (N29) wereanalyzed for the cAMP response to peptide stimulation. The subcloneswere split into 24 well plates. Cells were starved for 1 h in DMEMwithout FBS, then medium was replaced with 0.5 ml fresh DMEM (withoutFBS) with the compounds as indicated. In FIG. 26, neurons were exposedto 10⁻⁷ M (100 nM) TCAP peptide. All peptides were diluted in DMEMcontaining IBMX (100 μM). After a 15 min incubation at 37° C., 1 ml ofice-cold ethanol was added to each well. Cells were scraped from theplate and kept at −20° C. until the amounts of intracellular cAMP weredetermined in triplicate by RIA (Biotechnologies Inc., Stoughton, Mass.)according to the manufacturer's instructions.

B. Proteomic Profiling Using TCAP 3

NPY17 (N38) immortalized neurons were treated with 100 nM TCAP-3 andsubjected to proteomic profiling. In this procedure, the nuclei of cellsare isolated and the proteins extracted. This method provides anindication of proteins that are up or down regulated by a giventreatment. The proteomic profile indicated that the majority of proteinsup-regulated were associated with cell cycle, metabolism and the stressresponse. A number of cytoskeletal proteins were also upregulated. Thisobservation is of particular importance as many antidepressants havebeen shown to increase spine density and arborization of neuronalprocesses. Such events are regulated by cytoskeletal proteins.

Proteomic profiling Up regulated at 12 hours Protein ProcessingParvulin; protein chaperone Transcriptional Regulation Npw28 bindingprotein Staufen; mRNA targetting histone acetylmethyl transferasehelicase Cell Growth, Cycle and Proliferation MIDA1; cell growthregulator Smad 5; TGFbeta signalling STE20-like kinase; apoptosis Kp78,wnt pathway activation Integrin linked kinase 1, wnt pathway p53 targetprotein, tumor suppressor IGFBP, growth regulation esp1, cell divisionsepiapterin reductase TGFbeta Bp1, growth regulation Rad23, uv repairprotein Extracellular Matrix protocadherin gammaB5 talin Cytoskeletonalpha actinin4 CLP36, actinin4 interaction Cell Signalling PKC iota

B. MicroArray Studies I. Method RNA Isolation

Total RNA (TRNA) was isolated from 3 independent treated and untreatedN38 hypothalamic cell cultures, pooled (to reduce the noise), utilizingTrizol Reagent (GIBCO/BRL) following the manufacturer's protocol. Thequality of total RNA was assessed using an Agilent 2100 Bioanalyzer(version A.02.01S1232, Agilent Technologies). Only RNA with the OD ratioof 1.99-2.0 at 260/280 was used

Oligonucleotide Arrays (Hybridization, Staining, and Scanning)

Hybridizations were performed on the Mouse MU74Av2 GeneChip Set(Affymetrix, Santa Clara, Calif.). Samples were prepared forhybridization according to Affymetrix instructions. Briefly, a primerencoding the T7 RNA polymerase promoter linked to oligo-dT₁₇ was used toprime double-stranded cDNA synthesis from each mRNA sample usingSuperscript II RNase H⁻ reverse transcriptase (Life Technologies,Rockville, Md.). Each purified (Qiaquick kit, Qiagen) double-strandedcDNA was in vitro transcribed using T7 RNA polymerase (T7 kit; Enzo),incorporating biotin-UTP and biotin-CTP (Enzo Biochemicals, New York,N.Y.) into the cRNAs, followed by purification using RNEasy (Qiagen) andquantitated by measuring absorption at 260 nm/280 nm. Samples werefragmented and hybridized to the Chip for 16 h at 45° C. and scanned(GeneArray scanner, Affymetrix). MicroArray Suite Version 5 (MASv5;Affymetrix) was used to scale intensities across the Genechips to 150fluorescence units, and to determine expression values for each gene onthe chip. The expression value for each gene was determined bycalculating the average of differences (perfect match intensity minusmismatch intensity) of the probe pairs in use for the gene.

Data Analysis

Gene analysis software: Data analysis was performed using twoindependent softwares, GeneChip and GeneSpring. To identifydifferentially expressed transcripts, pairwise comparison analyses werecarried out with MicroArray Suite Version 5 MicroArray Suite Version 5(MASv5; Affymetrix). This approach, which is based on the Mann-Whitneypairwise comparison test, allows the ranking of results by concordance,as well as the calculation of significance (P value) of each identifiedchange in gene expression. Statistically significant genes (P<0.05) wereselected for further analysis. Moreover, statistically significantchanges in mean expression values were determined by importing the datafrom MASv5 into GeneSpring 5 (Silicon Genetics, Redwood City, Calif.). Astepwise process was followed, first with normalizations. A per-chipfollowed by a per-gene normalization in order to facilitate directcomparison of biological differences. Next, a second method of filterusing Affymetrix data and p value with cut-off of P<0.005 generated4,841 genes which were used for subsequent analysis utilizingHierarchical Clustering, k-means, Self Organization Map (SOM) utilizingGeneSpring 5.0.

II. Results

Further, to demonstrate that the cell lines can be used as a model forstudying TCAP responsiveness, modulation, and in screening for TCAPmodulators, microarray studies were performed on 1 nM TCAP-1 [SEQ ID NO5 plus amidation signal GRR at C-terminus] treated N38 hypothalmiccells, which do not possess either CRF receptor subtype (Table 4). RNAsisolated from treated and untreated cells were analyzed onoligonucleotide arrays representing 12,884 mouse genes (Affymetrix,http://www.affymetrix.com). Standard filtering (p<0.005) andhierarchical clustering algorithm (average linage method: GeneSpringsoftware—Silicon Genetics) identified significant changes in theexpression of 4, 841/12,885 genes with 166 genes showing 1.5 folddown-regulation and 35 genes up-regulation in the TCAP-1-treated cellscompared to the untreated cells. At 16 hours post-treatment, asignificant decrease occurred among several genes, notably, GAS5, SDPRand CD95 that have been associated with growth arrest or apoptoticevents (45-47). In contrast, upregulated genes including MK167, MOPS andGDAP10 have been associated with cell proliferation and cell cyclemodulation (48-50). A G-protein coupled receptor-related signaltransduction pathway is indicated by the regulation of genes, CREM,AKAP8, AKAP95 and PDE6A. Downstream effectors of RAS such as EFK1 andRGL were also down regulated. Downregulation of the A kinase anchoringprotein AKAP95 but upregulation of AKAP8 suggests that TCAP may act, inpart, by changing the targeting pattern of PKA (51). The upregulation ininducible nitric oxide (INOS), a intracellular voltage-gated chloridechannel (CLCN3) and the serotonin transporter (SLC6A4) may reflect thedown stream actions of cAMP-mediated signal cascade and indicates thepotential for TCAP to be involved in neuronal signaling systems. A rolein interneuron communication by TCAP is also indicated by the modulationof genes associated with the regulation of vesicle trafficking. Thus,the TCAP responsive cell lines can be used to screen for modulators ofneuronal function that affect growth, differentiation and communication.

SUMMARY OF EXPERIMENTAL RESULTS

The teneurin c-terminal associated peptide (TCAP) represents theterminal 40 to 41 residues on all four of the known teneurin (Ten M)proteins. On all four of the teneurins, TCAP shows the greatest sequencehomology among the entire exon suggesting that it is under the moststringent physiological constraints of the protein. TCAP is a potentinhibitor of neuronal and fibroblast growth possibly by arresting cellcycle. When injected into rat brain it increased the startle reflex anddecreased self-administered reward behaviour and was shown to modulatethe stress response. These data indicates that TCAP represents a novelneurohormonal system associated with neuronal growth and development.

The finding of a TCAP-like peptide on the carboxy terminus of a type IItransmembrane protein is unusual. Assuming that the protein is onlyexpressed on the extracellular face of the cell, then it is likely thatthe peptide acts in a paracrine manner to regulate the surroundingcells. All Ten M proteins possess a basic residue in positions −1 and −8upstream from the putative cleavage site from the peptide. Such a basicresidue arrangement is recognized by the prohormone convertase 7 (PC7)family of proteases (Saideh and Chretien, 1997), for the processing ofpeptide prohormones. Assuming this to be the case, then the requisitePC7-like protein would need to be expressed also on the extracellularface of the cell, or perhaps on the extracellular face of an adjacentcell. Alternatively, the protease may be secreted and act in a moremobile fashion. In any case, the release of the cleaved peptide wouldunlikely to occur in the bolus seen by vesicular release. It is alsoconceivable that the Ten-M protein is expressed in vesicles of theregulated pathway where intravesicular proteases could cleave thepeptide before exocytosis. However, the synthetic peptide shows a strongtendency to aggregate and precipitate at concentrations higher than 2ug/ul. This is likely due to the high number (15) of leucines,isoleucines, valine, tyrosines and phenylalanine within the peptide.Peptides that have high vesicular concentrations such as theurocortin-like peptide, sauvagine, found in the skin of a neotropicalfrog, Phyllomedusa sauvagei, tend to have a low proportion ofhydrophobic residues (Pallai et al., 1983). Thus this physicalcharacteristic of the TCAP peptide supports its preferential releasefrom the cleavage from the extracellular face of the plasma membrane.

The TCAP portion of the Ten-M proteins appears to be the most highlyconserved of the terminal exon of the protein. Such high levels ofconservation occur when there are many physiological, biochemicalconstraints acting upon the sequence to inhibit change. Such resistanceto change could result from essential interactions with processing ordegrading enzymes, receptors, and/or transport proteins. The level ofconservation of 90% between the paralogues in vertebrates is high incomparison to the CRF group of peptides to which TCAP appears to be mostclosely related.

In any case, a number of other bioactive peptides are initiallyexpressed and processed in the same manner as TCAP. Other bioactivepeptides such as tumor necrosis factor (TNF) (Utsumi et al., 1995),Apo-2 ligand (Pitti et al., 1996) and fractalkine (Garton et al., 2001)are processed in this manner. These peptides are directed outward at theend of the C-terminus on the extracellular face. Peptides processed andexpressed in this manner have the potential for a variety of endocrineor juxtacrine roles. For example they may act as an adhesion moleculefor cells displaying the appropriate receptor. Such actions could beparticularly important during the migration of neurons in the developingbrain, allowing neurons to be directed to a specific target.Alternatively, the peptide may be cleaved via a membrane-bound orextracellular matrix-associated protease to act as a paracrine/autocrinefactor to modulate the actions of surrounding cells. Such a mechanismwould be important for cells to protect against low oxygen stresseswhich occur in ischaemia. All three cytokines appear to be processed bya tumor necrosis factor alpha converting enzyme (TACE, ADAM17). Thisenzyme is also capable of cleaving the cell-surface ectodomain of theamyloid-beta precursor protein (Skovronsky et al. 2001), thus decreasingthe generation of amyloid beta suggesting it may have a role in theaetiology of Alzheimer's disease.

The TCAP peptide appears to regulate several physiological events. In amouse neuronal cell line, Gn11, and a rat fibroblast cell line, TGR1,treatment of TCAP at concentrations of 10⁻⁹ to 10⁻⁶ M could inhibitproliferation in a dose-dependent manner where maximal inhibition occursat about 60%. There was no evidence of apoptosis or necrosis of thecells and morphology did not differ between treated and untreated cells.

This stress-related studies indicate an ability of the TCAP peptide toinhibit the damage done by environmental stresses on cells that wouldoccur during periods of ischaemia or perhaps various neurodegenerativediseases. Given the decrease of proliferation rate seen in unstressedcells and the apparent increase in stressed cells suggests that TCAP maybe acting in part to reduce the metabolic activity of the cell. Otherrelated peptides have a similar effect. For example, urocortin canprevent cell death in primary cardiac myocyte cultures by stimulatingthe p42/p44 mitogen-activated protein (MAP) kinase pathway (Latchman,2001). Under stressful conditions such as heat shock (Okosi et al.,1998) or ischaemia (Brar et al., 1999), urocortin mRNA is upregulated incultured cardiac cells, and is also secreted into the medium (Brar etal., 1999), suggesting that it too, is acting in a paracrine fashion toregulate cell metabolism. This effect is much greater by urocortin thanCRF. This is of particular interest given that the urocortin paraloguesof the CRF family appear to represent evolutionarily older sequencesthan CRF (Lovejoy and Balment, 1999). Such paracrine actions on cellmetabolism may be then one of the initial and critical functions of theancestor gene that gave rise to both the TCAP and CRF/urocortin/diureticgroup of peptides.

The data obtained so far can be used to delineate a tentative model forthe mechanism for TCAP (FIG. 19). Initially, a stressor, such as changesin pH, temperature, or O₂ levels, or alternatively, a stress-inducedligand triggers an up-regulation of the Ten-M protein. Such stressorslikely act through a number of signal transduction pathways includingadenylate cyclase and guanylate cyclase. It is conceivable that thestressor also up-regulates the Ten-M cleaving enzyme such as TACE orPC7. The TCAP ligand is then cleaved from its protein and is free to actin an autocrine and paracrine manner. It binds to a G-protein coupledreceptor that subsequently interacts with a G-inhibitory protein. Thisinhibits cAMP and cGMP production to inhibit activation of the cell. Ina dividing neuron this would act to inhibit proliferation or migration,and in a mature non-dividing neuron could manifest as a reduction ofsynaptic output thereby inhibiting the neurological response of anactivated nucleus of cells in the brain.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Cell Lines Screening MARKER N-7 N-22 N-29 N-38 T antigen + + + +NSE + + + + GFAP − − − − NT − − − − ER alpha + + + + ER beta + + + +Tph + + − w Socs-3 + + + + AR − − N/A + G2R + + + − CRF − − − −GnRH + + + w POMC − + + − Gal + − w − Lep Receptor − + + w Agrp + + + +Cart − − − − NPY − − + + proGlu − w w − TH + − + − GHRH − + + + Avp + +w w proTRH − − − − Ucn − − − − MCH + N/A + + orexin − − − − DAT strong −w − CRFR1 − − − − CRFR2 − − − − Aromatase − − − strong GnRH Receptor − −− − Insulin receptor + + + + Oxytocin + + + + New-1 − − − − New-2 − − −− New-4 − − + − GHS-R N/A N/A N/A − Leptin som NTR + w N/A − mc3R mc4RN/A N/A N/A − NPY-Y1 NPY-Y2 CRLR N/A N/A N/A − Ghrelin + + N/A + Ghrelinvariant + − N/A − The following abbreviations will have their standardscientific abbreviations: T-Ag, Large T-antigen; NSE, neuron-specificenolase; GFAP, glial fibrillary acidic protein; SNTX, syntaxin; ER,estrogen receptor; AR, androgen receptor; LepR, leptin receptor b;Glp-2R (also G2R), glucagon-like peptide 2 receptor; SOCS-3, suppressorof cytokine signaling 3; NPY, neuropeptide Y; AGRP, agoutirelatedpeptide; POMC, proopiomelanocortin; CART, cocaine and amphetamineregulated transcript; MCH, melanin-concentrating hormone; Ucn,urocortin; NT, neurotensin; Gal, galanin; Orx, orexin; DAT, dopaminetransporter; CRFR, corticotrophin-releasing factor receptor; proGlu,proglucagon; GHRH, growth hormone-releasing hormone; GnRH,gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormonereceptor; CRF, corticotropin-releasing factor; TRH, thyroid-releasinghormone; AVP, arginine vasopressin; OXY, oxytocin; Arom, aromatase; TPH,tryptophan hydroxylase; TH, tyrosine hydroxylase; TenM-1 (also New-1);TenM-2 (also New-2); TenM-3 (also New-3); and TenM-4 (also New-4),Teneurins 1-4; GHS-R, growth hormone secratogue receptor; Lep, leptin;SOM, somatostatin; NTR, neurotensin receptor; MC3R, melanocortinreceptor-3; MC4R, melanocortin receptor-4; NPY-Y1, NPY receptor Y1;NPY-Y2, NPY receptor Y2; CRLR, calcitonin receptor like receptor; nd,not done; na, not done; w, weak expression.

TABLE 2 Genes Regulated by TCAP-1 at 16 hours Affimetrix Acc No. FoldCluster Gene Probe No. GB Function change Growth/ GAS5 98530 AI849615Growth arrest specific transcript 0.46 Differentiation SDPR 160373AI839175 Serum deprivation response protein 0.57 PPAN 160802 AA674812Peter Pan homologue 0.62 CD95 102921 M83649 Fas antigen 0.61 CRD-BP102627 AF061569 CRD-binding protein 0.59 SSG1 160298 AW122012 Steroidsensitive gene 1 0.62 DIP1/2 97353 AI837497 DAB2 interacting protein0.68 GBP3 103202 AW047476 Guanylate binding protein 0.63 P202 161173AV229143 202 interferon activatable protein 0.61 CAII 103441 AI94248Casein kinase II 0.61 INI1B 99924 AW121845 Integrase interacting protein1B 0.48 MMP1 100484 X66473 Matrix metalloproteinase 1 0.55 MMP10 94724Y13185 Matrix metalloproteinase 10 0.59 PTK7 92325 AI326889 Receptorprotein tyrosine kinase 1.53 P204 98466 M31419 Interferon activatableprotein 1.85 MKI67 161931 AV309347 Cell cycle protein regulator 1.70MOP3 102382 AB014494 Circadian rhythm regulator 1.57 ST7 160591 AI504013Suppressor of tumourigenicity 1.97 GDAP10 94192 Y17860 Gangliosideinduced diff. protein 10 1.62 Signalling/ ERK1 101834 Z14249 Mitogenactivated protein kinase 0.64 Communication ALK3 92767 D16250 Bonemorphogenic protein receptor 0.60 BMP4 93456 L47480 Bone morphogenicprotein-4 0.52 IL1R 93914 M20658 Interleukin 1 receptor 0.60 GR 98818X04435 Glucocorticoid receptor 0.66 BARK1 104270 AA982714 β adrenergicreceptor kinase 1 0.61 CAMIII 92631 M19380 Calmodulin III 0.53 PCDHγ160976 AA222943 protocadherin γ 0.42 AKAP95 95001 AB028920 A kinaseanchor protein 95 0.60 TTF-1IP 161019 W41560 TTF-1 interacting peptide0.50 CREMβ1 100533 M60285 cAMP-responsive element modulator 1.61 AKAP8161088 AV171460 A kinase anchor protein 8 1.58 PDE6A 100696 X60664 cGMPPhosphodiesterase α 1.68 INOS 104420 U43428 Inducible nitric oxidesynthetase 1.50 FNBX 92754 D49920 Ferredoxin-NADP reductase 1.61 SLC6A4161695 AV230927 Serotonin transporter 1.53 CLCN3 94465 AF029347 Chloridechannel protein 3 1.66 Processing ARF1 95156 AI1853873 ADP ribosylationfactor 1 0.63 CLM2-B 93492 AB013469 Cytohesin-2 0.63 YIPID 99675AI839766 Rab-mediated membrane transport 1.88 RAB10 160149 AI841543 Rasoncogene homologue 1.62 GP25L2 100074 AW046723 gp25L brings cargoforward from ER 1.53 AP4S1 104561 AI847561 Adaptor related proteincomplex 1.52

The change in expression levels is indicated relative to the untreatedcontrol cell for the same time period of 16 hours. Values >1.5 fold or<0.70 fold were considered significant.

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1. An isolated nucleic acid molecule encoding a teneurin c-terminalassociated peptide consisting of: (a) a nucleic acid sequence as shownin SEQ. ID. NOS.: 18-20, 25-28, 33-36, 41-44, 49-52, 57-60, 65-68,73-76, 81-84, 89-92, 97-100 or that wherein T can also be U or thatencodes a peptide having an amino acid sequence selected from the groupconsisting of: SEQ. ID. NOS: 13, 14, 21, 22, 29, 30, 37, 38, 45, 46, 53,54, 61, 62, 69, 70, 77, 78, 85, 86, 93, 94, 101, 103 or that further hasan amidation signal sequence, at the carboxy terminus of said peptides,or has SEQ. ID. NO. 15, 16, 23, 24, 31, 32, 39, 40, 47, 48, 55, 56, 63,64, 71, 72, 79, 80, 97, 88, 95, 96; (b) a nucleic acid sequence that iscomplimentary to a nucleic acid sequence of (a); (c) a nucleic acidsequence that has substantial sequence homology to a nucleic acidsequence of (a) or (b); (d) a nucleic acid sequence that is an analog ofa nucleic acid sequence of (a), (b) or (c); or (e) a nucleic acidsequence that hybridizes to a nucleic acid sequence of (a), (b), (c) or(d) under stringent hybridization conditions.
 2. A isolated nucleic acidmolecule of claim 1 wherein the amidation signal sequence is GKR or GRR.3. A nucleic acid molecule of claim 2 wherein the sequence is selectedfrom the group of sequences consisting of SEQ. ID. NOS:15, 16, 23, 24,31, 32, 39, 40, 47, 48, 55, 56, 63, 64, 71, 72, 79, 80, 97, 88, 95, 96.4. An isolated nucleic acid molecule encoding a TCAP peptide wherein thepeptide has neuronal communication activity and/or stress modulationactivity and/or cell proliferation inhibition activity.
 5. An antisenseoligonucleotide that is complimentary to a nucleic acid sequenceaccording to claims 1 to
 4. 6. An expression vector comprising a nucleicacid molecule of any one of claims 1 to
 5. 7. A host cell transformedwith an expression vector of claim
 6. 8. An isolated teneurin c-terminalassociated peptide which has the amino acid sequence as shown in SEQ.ID. NOS: 13, 14, 21, 22, 29, 30, 37, 38, 45, 46, 53, 54, 61, 62, 69, 70,77, 78, 85, 86, 93, 94, 101, 103 or a fragment, analog, homolog,derivative or mimetic thereof or a biologically active fragment thereof.9. An isolated teneurin c-terminal associated peptide of claim 8 furthercomprising an amidation signal sequence at the carboxy terminus.
 10. Ateneurin c-terminal associated peptide according to claim 8 or 9 whereinthe peptide has anxiogenic activity.
 11. An antibody that can bind apeptide according to any one of claims 8 to
 10. 12. A method ofidentifying substances which can bind with a teneurin c-terminalassociated peptide, comprising the steps of: (a) incubating a teneurinc-terminal associated peptide and a test substance, under conditionswhich allow for formation of a complex between the teneurin c-terminalassociated peptide and the test substance, and (b) assaying forcomplexes of the teneurin c-terminal associated peptide and the testsubstance, for free substance or for non complexed teneurin c-terminalassociated peptide, wherein the presence of complexes or reduced levelsas compared to a starting level of free substance or non-complexedteneurin c-terminal associated peptide indicates that the test substanceis capable of binding to the teneurin c-terminal associated peptide. 13.A method for identifying a compound that affects the activity orexpression of teneurin c-terminal associated peptide comprising: (a)incubating a test compound with a teneurin c-terminal associated peptideor a nucleic acid encoding a teneurin c-terminal associated peptide; and(b) determining an amount of teneurin c-terminal associated peptideprotein activity or expression and comparing with a control, wherein achange in the TCAP peptide activity or expression as compared to thecontrol indicates that the test compound has an effect on TCAP peptideactivity or expression.
 14. The method of claim 13 wherein in step (a) atest compound is incubated with a teneurin c-terminal associated peptideand teneurin c-terminal associated peptide substrate under conditionsthat permit interaction of the peptide and substrate, and step (b) andin step (b) the peptide activity on the substrate is determined.
 15. Themethod of claim 13, wherein in step (a) a cell expressing a teneurinc-terminal associated peptide and activity, is incubated with a testcompound, under conditions where teneruin c-terminal associated peptideis active and in step (b) teneurin c-terminal associated peptideactivity is determined.
 16. The method of claim 15, wherein the teneurinc-terminal associated peptide activity is determined by detecting thelevels of cAMP and cGMP before and after incubation with the testcompound, or as compared to a control, wherein a change in magnitude oflevels of cAMP or cGMP as compared to a baseline or control level isindicative that the test compound is a modulator of teneurin c-terminalassociated peptide activity.
 17. The method of claim 16, wherein thereduction of cAMP or cGMP in the presence of a test compound is lessthan in the control or baseline level or is greater than in the controlor baseline level of TCAP activity indicates that the test compound isan inhibitor of c-teneurin associated peptide activity.
 18. A method ofidentifying a compound that affects the regulation of neuronal growthcomprising: (a) incubating a test compound with a teneurin c-terminalassociated peptide or a nucleic acid encoding a teneurin c-terminalassociated peptide; and (b) determining an amount of teneurin c-terminalassociated peptide protein activity or expression and comparing with acontrol, wherein a change in the TCAP peptide activity or expression ascompared to the control indicates that the test compound has an effecton the regulation of neuronal growth.
 19. A method of inhibiting cellproliferation comprising administering to a cell, an effective amount ofteneurin c-terminal associated peptide that inhibits cell proliferation.20. A method according to claim 19 wherein the cell is selected from thegroup consisting of neuronal or fibroblast cells.
 21. A method ofdetecting a condition associated with the aberrant regulation ofneuronal growth comprising assaying a sample for (a) a nucleic acidmolecule encoding a teneurin c-terminal associated peptide or a fragmentthereof or (b) a teneurin c-terminal associated peptide or a fragmentthereof.
 22. A method of treating a condition associated with theaberrant regulation of neuronal growth comprising administering to acell or animal in need thereof, an effective amount of a teneurinc-terminal associated peptide or an agent that modulates teneurinc-terminal associated peptide expression and/or activity.
 23. A methodaccording to claim 22 wherein the agent is selected from the groupconsisting of: a nucleic acid molecule encoding teneurin c-terminalassociated peptide; teneurin c-terminal associated peptide as well asfragments, analogs, derivatives or homologs thereof; antibodies;antisense nucleic acids; peptide mimetics; and substances isolated usingthe screening methods described in claims 12-20.
 24. A method ofinducing an anxiogenic response in a subject comprising administering toa subject an effective amount of teneurin c-terminal associated peptideto induce an anxiogencic response.
 25. A method of inhibiting ananxiogenic response in a subject comprising administering to a subjectan effective amount of an inhibitor of teneruin c-terminal associatedpeptide to inhibit an anxiogenic response.
 26. A method of claim 25wherein the inhibitor is identified according to the method of any oneof claims 13 to
 18. 27. A method of inhibiting the damage caused byphysiological stresses comprising administering to a cell, an effectiveamount of teneurin c-terminal associated peptide that protects cellsfrom the physiological stresses.
 28. A method of modulating the stressresponse in an animal comprising administering an effective amount ofTCAP to said animal.
 29. A method of modulating anxiety response in ananimal comprising administering an effective amount of TCAP to saidanimal.
 30. The method of increasing anxiety in a low anxiety animalcomprising administering to said animal an effective amount of TCAP. 31.A method of decreasing anxiety in a high anxiety animal comprisingadministering to said animal an effective amount of TCAP.
 32. A methodof normalizing anxiety response in an animal comprising administering tosaid animal an effective amount of TCAP.
 33. A method of treating cancerin an animal comprising administering an effective amount of TCAP tosaid animal.
 34. A pharmaceutical composition comprising TCAP and apharmaceutically acceptable vehicle.